Virion-like delivery particles for self-replicating RNA molecules

ABSTRACT

Nucleic acid immunisation is achieved by delivering a self-replicating RNA encapsulated within a small particle. The RNA encodes an immunogen of interest, and the particle may deliver this RNA by mimicking the delivery function of a natural RNA virus. Thus the invention provides a non-virion particle for in vivo delivery of RNA to a vertebrate cell, wherein the particle comprises a delivery material encapsulating a self-replicating RNA molecule which encodes an immunogen. These particles are useful as components in pharmaceutical compositions for immunising subjects against various diseases.

This application is the U.S. National Phase of International ApplicationNo. PCT/US2011/043103, filed Jul. 6, 2011 and published in English,which claims the benefit of US Provisional Application No. 61/361,828,filed Jul. 6, 2010, the complete contents of which are herebyincorporated herein by reference for all purposes.

TECHNICAL FIELD

This invention is in the field of non-viral delivery of self-replicatingRNAs for immunisation.

BACKGROUND ART

The delivery of nucleic acids for immunising animals has been a goal forseveral years. Various approaches have been tested, including the use ofDNA or RNA, of viral or non-viral delivery vehicles (or even no deliveryvehicle, in a “naked” vaccine), of replicating or non-replicatingvectors, or of viral or non-viral vectors.

There remains a need for further and improved nucleic acid vaccines and,in particular, for improved ways of delivering nucleic acid vaccines.

DISCLOSURE OF THE INVENTION

According to the invention, nucleic acid immunisation is achieved bydelivering a self-replicating RNA encapsulated within and/or adsorbed toa small particle. The RNA encodes an immunogen of interest, and theparticle may deliver this RNA by mimicking the delivery function of anatural virus.

Thus the invention provides a non-virion particle for in vivo deliveryof RNA to a vertebrate cell, wherein the particle comprises a deliverymaterial encapsulating a self-replicating RNA molecule which encodes animmunogen. The invention also provides a non-virion particle for in vivodelivery of RNA to a vertebrate cell, wherein the particle comprises adelivery material on which a self-replicating RNA molecule which encodesan immunogen is adsorbed. These particles are useful as components inpharmaceutical compositions for immunising subjects against variousdiseases. The combination of utilising a non-virion particle to delivera self-replicating RNA provides a way to elicit a strong and specificimmune response against the immunogen while delivering only a low doseof RNA. Moreover, these particles can readily be manufactured at acommercial scale.

The Particle

Particles of the invention are non-virion particles i.e. they are not avirion. Thus the particle does not comprise a protein capsid. Byavoiding the need to create a capsid particle, the invention does notrequire a packaging cell line, thus permitting easier up-scaling forcommercial production and minimising the risk that dangerous infectiousviruses will inadvertently be produced.

Instead of encapsulating RNA in a virion, particles of the invention areformed from a delivery material. Various materials are suitable forforming particles which can deliver RNA to a vertebrate cell in vivo.Two delivery materials of particular interest are (i) amphiphilic lipidswhich can form liposomes and (ii) non-toxic and biodegradable polymerswhich can form microparticles. Where delivery is by liposome, RNA shouldbe encapsulated; where delivery is by polymeric microparticle, RNA canbe encapsulated or adsorbed. A third delivery material of interest isthe particulate reaction product of a polymer, a crosslinker, a RNA, anda charged monomer.

Thus one embodiment of a particle of the invention comprises a liposomeencapsulating a self-replicating RNA molecule which encodes animmunogen, whereas another embodiment comprises a polymericmicroparticle encapsulating a self-replicating RNA molecule whichencodes an immunogen, and another embodiment comprises a polymericmicroparticle on which a self-replicating RNA molecule which encodes animmunogen is adsorbed. In all three cases the particles preferably aresubstantially spherical. In a fourth embodiment a particle of theinvention comprises the particulate reaction product of a polymer, acrosslinker, self-replicating RNA molecule which encodes an immunogen,and a charged monomer. These particles are formed within molds and socan be created with any shape including, but not limited to, spheres.

RNA can be encapsulated within the particles (particularly if theparticle is a liposome). This means that RNA inside the particles is (asin a natural virus) separated from any external medium by the deliverymaterial, and encapsulation has been found to protect RNA from RNasedigestion. Encapsulation can take various forms. For example, in someembodiments (as in a unilamellar liposome) the delivery material forms aouter layer around an aqueous RNA-containing core, whereas in otherembodiments (e.g. in molded particles) the delivery material forms amatrix within which RNA is embedded. The particles can include someexternal RNA (e.g. on the surface of the particles), but at least halfof the RNA (and ideally all of it) is encapsulated. Encapsulation withinliposomes is distinct from, for instance, the lipid/RNA complexesdisclosed in reference 1.

RNA can be adsorbed to the particles (particularly if the particle is apolymeric microparticle). This means that RNA is not separated from anyexternal medium by the delivery material, unlike the RNA genome of anatural virus. The particles can include some encapsulated RNA (e.g. inthe core of a particle), but at least half of the RNA (and ideally allof it) is adsorbed.

Liposomes

Various amphiphilic lipids can form bilayers in an aqueous environmentto encapsulate a RNA-containing aqueous core as a liposome. These lipidscan have an anionic, cationic or zwitterionic hydrophilic head group.Formation of liposomes from anionic phospholipids dates back to the1960s, and cationic liposome-forming lipids have been studied since the1990s. Some phospholipids are anionic whereas other are zwitterionic andothers are cationic. Suitable classes of phospholipid include, but arenot limited to, phosphatidylethanolamines, phosphatidylcholines,phosphatidylserines, and phosphatidyl-glycerols, and some usefulphospholipids are listed in Table 1. Useful cationic lipids include, butare not limited to, dioleoyl trimethylammonium propane (DOTAP),1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterioniclipids include, but are not limited to, acyl zwitterionic lipids andether zwitterionic lipids. Examples of useful zwitterionic lipids areDPPC, DOPC and dodecylphosphocholine. The lipids can be saturated orunsaturated. The use of at least one unsaturated lipid for preparingliposomes is preferred. If an unsaturated lipid has two tails, bothtails can be unsaturated, or it can have one saturated tail and oneunsaturated tail.

Liposomal particles of the invention can be formed from a single lipidor from a mixture of lipids. A mixture may comprise (i) a mixture ofanionic lipids (ii) a mixture of cationic lipids (iii) a mixture ofzwitterionic lipids (iv) a mixture of anionic lipids and cationic lipids(v) a mixture of anionic lipids and zwitterionic lipids (vi) a mixtureof zwitterionic lipids and cationic lipids or (vii) a mixture of anioniclipids, cationic lipids and zwitterionic lipids. Similarly, a mixturemay comprise both saturated and unsaturated lipids. For example, amixture may comprise DSPC (zwitterionic, saturated), DlinDMA (cationic,unsaturated), and/or DMG (anionic, saturated). Where a mixture of lipidsis used, not all of the component lipids in the mixture need to beamphiphilic e.g. one or more amphiphilic lipids can be mixed withcholesterol.

The hydrophilic portion of a lipid can be PEGylated (i.e. modified bycovalent attachment of a polyethylene glycol). This modification canincrease stability and prevent non-specific adsorption of the liposomes.For instance, lipids can be conjugated to PEG using techniques such asthose disclosed in reference 2 and 3. Various lengths of PEG can be usede.g. between 0.5-8 kDa.

A mixture of DSPC, DlinDMA, PEG-DMG and cholesterol is used in theexamples.

Liposomal particles are usually divided into three groups: multilamellarvesicles (MLV); small unilamellar vesicles (SUV); and large unilamellarvesicles (LUV). MLVs have multiple bilayers in each vesicle, formingseveral separate aqueous compartments. SUVs and LUVs have a singlebilayer encapsulating an aqueous core; SUVs typically have a diameter≤50 nm, and LUVs have a diameter >50 nm. Liposomal particles of theinvention are ideally LUVs with a diameter in the range of 50-220 nm.For a composition comprising a population of LUVs with differentdiameters: (i) at least 80% by number should have diameters in the rangeof 20-220 nm, (ii) the average diameter (Zav, by intensity) of thepopulation is ideally in the range of 40-200 nm, and/or (iii) thediameters should have a polydispersity index <0.2. The liposome/RNAcomplexes of reference 1 are expected to have a diameter in the range of600-800 nm and to have a high polydispersity.

Techniques for preparing suitable liposomes are well known in the arte.g. see references 4 to 6. One useful method is described in reference7 and involves mixing (i) an ethanolic solution of the lipids (ii) anaqueous solution of the nucleic acid and (iii) buffer, followed bymixing, equilibration, dilution and purification. Preferred liposomes ofthe invention are obtainable by this mixing process.

Polymeric Microparticles

Various polymers can form microparticles to encapsulate or adsorb RNAaccording to the invention. The use of a substantially non-toxic polymermeans that a recipient can safely receive the particles, and the use ofa biodegradable polymer means that the particles can be metabolisedafter delivery to avoid long-term persistence. Useful polymers are alsosterilisable, to assist in preparing pharmaceutical grade formulations.

Suitable non-toxic and biodegradable polymers include, but are notlimited to, poly(α-hydroxy acids), polyhydroxy butyric acids,polylactones (including polycaprolactones), polydioxanones,polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates,tyrosine-derived polycarbonates or polyester-amides, and combinationsthereof.

In some embodiments, the microparticles are formed from poly(α-hydroxyacids), such as a poly(lactides) (“PLA”), copolymers of lactide andglycolide such as a poly(D,L-lactide-co-glycolide) (“PLG”), andcopolymers of D,L-lactide and caprolactone. Useful PLG polymers includethose having a lactide/glycolide molar ratio ranging, for example, from20:80 to 80:20 e.g. 25:75, 40:60, 45:55, 50:50, 55:45, 60:40, 75:25.Useful PLG polymers include those having a molecular weight between, forexample, 5,000-200,000 Da e.g. between 10,000-100,000, 20,000-70,000,30,000-40,000, 40,000-50,000 Da.

The microparticles ideally have a diameter in the range of 0.02 μm to 8μm. For a composition comprising a population of microparticles withdifferent diameters at least 80% by number should have diameters in therange of 0.03-7 μm.

Techniques for preparing suitable microparticles are well known in theart e.g. see references 6, 8 (in particular chapter 7) and 9. Tofacilitate adsorption of RNA, a microparticle may include a cationicsurfactant and/or lipid e.g. as disclosed in references 10 & 11.

Microparticles of the invention can have a zeta potential of between40-100 mV.

One advantage of microparticles over liposomes is that they are readilylyophilised for stable storage.

Molded Particles

A third delivery material of interest is the particulate reactionproduct of a polymer, a crosslinker, a self-replicating RNA whichencodes an immunogen, and a charged monomer. These four components canbe mixed as a liquid, placed in a mold (e.g. comprising aperfluoropolyether), and then cured to form the particles according tothe mold's shape and dimensions. Details of a suitable production methodare disclosed in ref. 12. These methods provide a biodegradablecrosslinked oligomeric polymer nanoparticle.

Ideally the particles have a largest cross-sectional dimension of ≤5 μm.They may have an overall positive charge.

Suitable polymers include, but are not limited to: a poly(acrylic acid);a poly(styrene sulfonate); a carboxymethylcellulose (CMC); a poly(vinylalcohol); a poly(ethylene oxide); a poly(vinyl pyrrolidone); a dextran;a poly(vinylpyrolidone-co-vinyl acetate-co-vinyl alcohol). A preferredpolymer is a poly(vinyl pyrrolidinone). The amount of polymer forforming the particles can be between 2-75 wt % e.g. 10-60 wt %, 20-60 wt%.

Suitable crosslinkers can include a disulfide and/or ketal. For example,the crosslinker can comprise poly(epsilon-caprolactone)-b-tetraethyleneglycol-b-poly(epsilon-capro lactone)dimethacrylate,poly(epsilon-caprolactone)-b-poly(ethylene glycol)-b-poly(epsilon-caprolactone)dimethacrylate, poly(lactic acid)-b-tetraethyleneglycol-b-poly(lactic acid)dimethacrylate, poly(lacticacid)-b-poly(ethylene glycol)-b-poly(lactic acid)dimethacrylate,poly(glycolic acid)-b-tetraethylene glycol-b-poly(glycolicacid)dimethacrylate, poly(gly colic acid)-b-poly(ethyleneglycol)-b-poly(gly colic acid)dimethacrylate,poly(epsilon-caprolactone)-b-tetraethyleneglycol-b-poly(epsilon-caprolactone)diacrylate,poly(epsilon-caprolactone)-b-poly(ethyleneglycol)-b-poly(epsilon-caprolactone)diacrylate, poly(lacticacid)-b-tetraethylene glycol-b-poly(lactic acid)diacrylate, poly(lacticacid)-b-poly(ethylene glycol)-b-poly(lactic acid)diacrylate,poly(glycolic acid)-b-tetraethylene glycol-b-poly(glycolicacid)diacrylate, poly(glycolic acid)-b-poly(ethyleneglycol)-b-poly(glycolic acid)diacrylate, silane, silicon containingmethacrylates, or dimethyldi(methacryloyloxy-1-ethoxy)silane. The amountof crosslinker for forming the particles can be between 10-25 wt % e.g.10-60 wt %, 20-60 wt %.

Charged monomers can be cationic or anionic. These include, but are notlimited to: [2-(acryloyloxy)ethyl]trimethyl ammonium chloride (AETMAC)and 2-aminoethyl methacrylate hydrochloride (AEM-HCl). The amount ofcharged monomer for forming the particles can be between 2-75 wt %.

The amount of RNA for forming the particles can be between 0.25-20 wt %.

A pre-cure mixture inside a mold can include an initiator. For instance,the mold can include ≤1 wt % initiator, ≤0.5 wt % initiator, or ≤0.1 wt% initiator. Between 0.1-0.5% initiator is useful. Photoinitiators suchas DEAP and DPT are useful e.g. for use with ultraviolet curing.

The invention can use any of the materials disclosed in Table 1 orExamples 1-15 of reference 12, except that the siRNA components thereinwill be replaced by self-replicating RNAs as herein.

The RNA

Particles of the invention include a self-replicating RNA molecule which(unlike siRNA) encodes an immunogen. After in vivo administration of theparticles, RNA is released from the particles and is translated inside acell to provide the immunogen in situ.

Unlike reference 13, the RNA in particles of the invention isself-replicating. A self-replicating RNA molecule (replicon) can, whendelivered to a vertebrate cell even without any proteins, lead to theproduction of multiple daughter RNAs by transcription from itself (viaan antisense copy which it generates from itself). A self-replicatingRNA molecule is thus typically a +-strand molecule which can be directlytranslated after delivery to a cell, and this translation provides aRNA-dependent RNA polymerase which then produces both antisense andsense transcripts from the delivered RNA. Thus the delivered RNA leadsto the production of multiple daughter RNAs. These daughter RNAs, aswell as collinear subgenomic transcripts, may be translated themselvesto provide in situ expression of an encoded immunogen, or may betranscribed to provide further transcripts with the same sense as thedelivered RNA which are translated to provide in situ expression of theimmunogen. The overall results of this sequence of transcriptions is ahuge amplification in the number of the introduced replicon RNAs and sothe encoded immunogen becomes a major polypeptide product of the cells.

One suitable system for achieving self-replication in this manner is touse an alphavirus-based replicon. These replicons are +-stranded RNAswhich lead to translation of a replicase (or replicase-transcriptase)after delivery to a cell. The replicase is translated as a polyproteinwhich auto-cleaves to provide a replication complex which createsgenomic −-strand copies of the +-strand delivered RNA. These −-strandtranscripts can themselves be transcribed to give further copies of the+-stranded parent RNA and also to give a subgenomic transcript whichencodes the immunogen. Translation of the subgenomic transcript thusleads to in situ expression of the immunogen by the infected cell.Suitable alphavirus replicons can use a replicase from a Sindbis virus,a Semliki forest virus, an eastern equine encephalitis virus, aVenezuelan equine encephalitis virus, etc. Mutant or wild-type virussequences can be used e.g. the attenuated TC83 mutant of VEEV has beenused in replicons [14].

A preferred self-replicating RNA molecule thus encodes (i) aRNA-dependent RNA polymerase which can transcribe RNA from theself-replicating RNA molecule and (ii) an immunogen. The polymerase canbe an alphavirus replicase e.g. comprising one or more of alphavirusproteins nsP1, nsP2, nsP3 and nsP4.

Whereas natural alphavirus genomes encode structural virion proteins inaddition to the non-structural replicase polyprotein, it is preferredthat the self-replicating RNA molecules of the invention do not encodealphavirus structural proteins. Thus a preferred self-replicating RNAcan lead to the production of genomic RNA copies of itself in a cell,but not to the production of RNA-containing virions. The inability toproduce these virions means that, unlike a wild-type alphavirus, theself-replicating RNA molecule cannot perpetuate itself in infectiousform. The alphavirus structural proteins which are necessary forperpetuation in wild-type viruses are absent from self-replicating RNAsof the invention and their place is taken by gene(s) encoding theimmunogen of interest, such that the subgenomic transcript encodes theimmunogen rather than the structural alphavirus virion proteins.

Thus a self-replicating RNA molecule useful with the invention may havetwo open reading frames. The first (5′) open reading frame encodes areplicase; the second (3′) open reading frame encodes an immunogen. Insome embodiments the RNA may have additional (e.g. downstream) openreading frames e.g. to encode further immunogens (see below) or toencode accessory polypeptides.

A preferred self-replicating RNA molecule has a 5′ cap (e.g. a7-methylguanosine). This cap can enhance in vivo translation of the RNA.In some embodiments the 5′ sequence of the self-replicating RNA moleculemust be selected to ensure compatibility with the encoded replicase.

A self-replicating RNA molecule may have a 3′ poly-A tail. It may alsoinclude a poly-A polymerase recognition sequence (e.g. AAUAAA) near its3′ end.

Self-replicating RNA molecules can have various lengths but they aretypically 5000-25000 nucleotides long e.g. 8000-15000 nucleotides, or9000-12000 nucleotides. Thus the RNA is longer than seen in siRNAdelivery.

Self-replicating RNA molecules will typically be single-stranded.Single-stranded RNAs can generally initiate an adjuvant effect bybinding to TLR7, TLR8, RNA helicases and/or PKR. RNA delivered indouble-stranded form (dsRNA) can bind to TLR3, and this receptor canalso be triggered by dsRNA which is formed either during replication ofa single-stranded RNA or within the secondary structure of asingle-stranded RNA.

The self-replicating RNA can conveniently be prepared by in vitrotranscription (IVT). IVT can use a (cDNA) template created andpropagated in plasmid form in bacteria, or created synthetically (forexample by gene synthesis and/or polymerase chain-reaction (PCR)engineering methods). For instance, a DNA-dependent RNA polymerase (suchas the bacteriophage T7, T3 or SP6 RNA polymerases) can be used totranscribe the self-replicating RNA from a DNA template. Appropriatecapping and poly-A addition reactions can be used as required (althoughthe replicon's poly-A is usually encoded within the DNA template). TheseRNA polymerases can have stringent requirements for the transcribed 5′nucleotide(s) and in some embodiments these requirements must be matchedwith the requirements of the encoded replicase, to ensure that theIVT-transcribed RNA can function efficiently as a substrate for itsself-encoded replicase.

As discussed in reference 15, the self-replicating RNA can include (inaddition to any 5′ cap structure) one or more nucleotides having amodified nucleobase. Thus the RNA can comprise m5C (5-methylcytidine),m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um(2′-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine);Am (2′-O-methyladenosine); ms2 m6A (2-methylthio-N6-methyladenosine);i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine);ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine);ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6.-hydroxynorvalylcarbamoyl adenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m11 (1-methylinosine);m′Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm(2T-O-methylcytidine); s2C (2-thiocytidine); ac4C(N4-acetylcytidine);f5C (5-fonnylcytidine); m5 Cm (5,2-O-dimethylcytidine); ac4 Cm(N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine);m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm(2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm(N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-β-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q(queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi(7-aminomethyl-7-deazaguanosine); G (archaeosine); D (dihydrouridine);m5Um (5,2′-β-dimethyluridine); s4U (4-thiouridine); m5s2U(5-methyl-2-thiouridine); s2Um (2-thio-2′-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U(5-carboxymethylaminomethyluridine); cnmm5Um(5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tm (2′-O-methylinosine);m4C(N4-methylcytidine); m4 Cm (N4,2-O-dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U(5-carboxymethyluridine); m6 Am (N6,T-O-dimethyladenosine); rn62 Am(N6,N6,O-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine); m2′2′7G(N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D(5-methyldihydrouridine); f5 Cm (5-formyl-2′-O-methylcytidine); m1Gm(1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyl adenosine)irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14(4-demethyl guanosine); imG2 (isoguanosine); or ac6A(N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine,7-substituted derivatives thereof, dihydrouracil, pseudouracil,2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil,5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil,5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine,5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine,5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine,8-azaguanine, 7-deaza-7-substituted guanine,7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine,8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine,2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine,8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine,7-deaza-8-substituted purine, or an abasic nucleotide. For instance, aself-replicating RNA can include one or more modified pyrimidinenucleobases, such as pseudouridine and/or 5-methylcytosine residues. Insome embodiments, however, the RNA includes no modified nucleobases, andmay include no modified nucleotides i.e. all of the nucleotides in theRNA are standard A, C, G and U ribonucleotides (except for any 5′ capstructure, which may include a 7′-methylguanosine). In otherembodiments, the RNA may include a 5′ cap comprising a7′-methylguanosine, and the first 1, 2 or 3 5′ ribonucleotides may bemethylated at the 2′ position of the ribose.

A RNA used with the invention ideally includes only phosphodiesterlinkages between nucleosides, but in some embodiments it can containphosphoramidate, phosphorothioate, and/or methylphosphonate linkages.

The amount of RNA per particle can vary, and the number of individualself-replicating RNA molecules per particle can depend on thecharacteristics of the particle being used. In general, a particle mayinclude from 1-500 RNA molecules. For a liposome the number of RNAmolecules is typically ≤50 per liposome e.g. <20, <10, <5, or 1-4. For apolymeric microparticle the number of RNA molecules will depend on theparticle diameter but may be ≤50 per particle (e.g. <20, <10, <5, or1-4) or from 50-200 per particle. Ideally, a particle includes fewerthan 10 different species of RNA e.g. 5, 4, 3, or 2 different species;most preferably, a particle includes a single RNA species i.e. all RNAmolecules in the particle have the same sequence and same length.

The Immunogen

Self-replicating RNA molecules used with the invention encode apolypeptide immunogen. After administration of the particles theimmunogen is translated in vivo and can elicit an immune response in therecipient. The immunogen may elicit an immune response against abacterium, a virus, a fungus or a parasite (or, in some embodiments,against an allergen; and in other embodiments, against a tumor antigen).The immune response may comprise an antibody response (usually includingIgG) and/or a cell-mediated immune response. The polypeptide immunogenwill typically elicit an immune response which recognises thecorresponding bacterial, viral, fungal or parasite (or allergen ortumour) polypeptide, but in some embodiments the polypeptide may act asa mimotope to elicit an immune response which recognises a bacterial,viral, fungal or parasite saccharide. The immunogen will typically be asurface polypeptide e.g. an adhesin, a hemagglutinin, an envelopeglycoprotein, a spike glycoprotein, etc.

Self-replicating RNA molecules can encode a single polypeptide immunogenor multiple polypeptides. Multiple immunogens can be presented as asingle polypeptide immunogen (fusion polypeptide) or as separatepolypeptides. If immunogens are expressed as separate polypeptides thenone or more of these may be provided with an upstream IRES or anadditional viral promoter element. Alternatively, multiple immunogensmay be expressed from a polyprotein that encodes individual immunogensfused to a short autocatalytic protease (e.g. foot-and-mouth diseasevirus 2A protein), or as inteins.

Unlike references 1 and 16, the RNA encodes an immunogen. For theavoidance of doubt, the invention does not encompass RNA which encodes afirefly luciferase or which encodes a fusion protein of E. coliβ-galactosidase or which encodes a green fluorescent protein (GFP).Also, the RNA is not total mouse thymus RNA.

In some embodiments the immunogen elicits an immune response against oneof these bacteria:

-   -   Neisseria meningitidis: useful immunogens include, but are not        limited to, membrane proteins such as adhesins,        autotransporters, toxins, iron acquisition proteins, and factor        H binding protein. A combination of three useful polypeptides is        disclosed in reference 17.    -   Streptococcus pneumoniae: useful polypeptide immunogens are        disclosed in reference 18. These include, but are not limited        to, the RrgB pilus subunit, the beta-N-acetyl-hexosaminidase        precursor (spr0057), spr0096, General stress protein GSP-781        (spr2021, SP2216), serine/threonine kinase StkP (SP1732), and        pneumococcal surface adhesin PsaA.    -   Streptococcus pyogenes: useful immunogens include, but are not        limited to, the polypeptides disclosed in references 19 and 20.    -   Moraxella catarrhalis.    -   Bordetella pertussis: Useful pertussis immunogens include, but        are not limited to, pertussis toxin or toxoid (PT), filamentous        haemagglutinin (FHA), pertactin, and agglutinogens 2 and 3.    -   Staphylococcus aureus: Useful immunogens include, but are not        limited to, the polypeptides disclosed in reference 21, such as        a hemolysin, esxA, esxB, ferrichrome-binding protein (sta006)        and/or the sta011 lipoprotein.    -   Clostridium tetani: the typical immunogen is tetanus toxoid.    -   Cornynebacterium diphtheriae: the typical immunogen is        diphtheria toxoid.    -   Haemophilus influenzae: Useful immunogens include, but are not        limited to, the polypeptides disclosed in references 22 and 23.    -   Pseudomonas aeruginosa    -   Streptococcus agalactiae: useful immunogens include, but are not        limited to, the polypeptides disclosed in reference 19.    -   Chlamydia trachomatis: Useful immunogens include, but are not        limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12,        OmcA, AtoS, CT547, Eno, HtrA and MurG (e.g. as disclosed in        reference 24. LcrE [25] and HtrA [26] are two preferred        immunogens.    -   Chlamydia pneumoniae: Useful immunogens include, but are not        limited to, the polypeptides disclosed in reference 27.    -   Helicobacter pylori: Useful immunogens include, but are not        limited to, CagA, VacA, NAP, and/or urease [28].    -   Escherichia coli: Useful immunogens include, but are not limited        to, immunogens derived from enterotoxigenic E. coli (ETEC),        enteroaggregative E. coli (EAggEC), diffusely adhering E. coli        (DAEC), enteropathogenic E. coli (EPEC), extraintestinal        pathogenic E. coli (ExPEC) and/or enterohemorrhagic E. coli        (EHEC). ExPEC strains include uropathogenic E. coli (UPEC) and        meningitis/sepsis-associated E. coli (MNEC). Useful UPEC        polypeptide immunogens are disclosed in references 29 and 30.        Useful MNEC immunogens are disclosed in reference 31. A useful        immunogen for several E. coli types is AcfD [32].    -   Bacillus anthracia    -   Yersinia pestis: Useful immunogens include, but are not limited        to, those disclosed in references 33 and 34.    -   Staphylococcus epidermis    -   Clostridium perfringens or Clostridium botulinums    -   Legionella pneumophila    -   Coxiella burnetii    -   Brucella, such as B. abortus, B. canis, B. melitensis, B.        neotomae, B. ovis, B. suis, B. pinnipediae.    -   Francisella, such as F. novicida, F. philomiragia, F.        tularensis.    -   Neisseria gonorrhoeae    -   Treponema pallidum    -   Haemophilus ducreyi    -   Enterococcus faecalis or Enterococcus faecium    -   Staphylococcus saprophyticus    -   Yersinia enterocolitica    -   Mycobacterium tuberculosis    -   Rickettsia    -   Listeria monocytogenes    -   Vibrio cholerae    -   Salmonella typhi    -   Borrelia burgdorferi    -   Porphyromonas gingivalis    -   Klebsiella

In some embodiments the immunogen elicits an immune response against oneof these viruses:

-   -   Orthomyxovirus: Useful immunogens can be from an influenza A, B        or C virus, such as the hemagglutinin, neuraminidase or matrix        M2 proteins. Where the immunogen is an influenza A virus        hemagglutinin it may be from any subtype e.g. H1, H2, H3, H4,        H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.    -   Paramyxoviridae viruses: Viral immunogens include, but are not        limited to, those derived from Pneumoviruses (e.g. respiratory        syncytial virus, RSV), Rubulaviruses (e.g. mumps virus),        Paramyxoviruses (e.g. parainfluenza virus), Metapneumoviruses        and Morbilliviruses (e.g. measles).    -   Poxyiridae: Viral immunogens include, but are not limited to,        those derived from Orthopoxvirus such as Variola vera, including        but not limited to, Variola major and Variola minor.    -   Picornavirus: Viral immunogens include, but are not limited to,        those derived from Picornaviruses, such as Enteroviruses,        Rhinoviruses, Heparnavirus, Cardioviruses and Aphthoviruses. In        one embodiment, the enterovirus is a poliovirus e.g. a type 1,        type 2 and/or type 3 poliovirus. In another embodiment, the        enterovirus is an EV71 enterovirus. In another embodiment, the        enterovirus is a coxsackie A or B virus.    -   Bunyavirus: Viral immunogens include, but are not limited to,        those derived from an Orthobunyavirus, such as California        encephalitis virus, a Phlebovirus, such as Rift Valley Fever        virus, or a Nairovirus, such as Crimean-Congo hemorrhagic fever        virus.    -   Heparnavirus: Viral immunogens include, but are not limited to,        those derived from a Heparnavirus, such as hepatitis A virus        (HAV).    -   Filovirus: Viral immunogens include, but are not limited to,        those derived from a Filovirus, such as an Ebola virus        (including a Zaire, Ivory Coast, Reston or Sudan ebolavirus) or        a Marburg virus.    -   Togavirus: Viral immunogens include, but are not limited to,        those derived from a Togavirus, such as a Rubivirus, an        Alphavirus, or an Arterivirus. This includes rubella virus.    -   Flavivirus: Viral immunogens include, but are not limited to,        those derived from a Flavivirus, such as Tick-borne encephalitis        (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever        virus, Japanese encephalitis virus, Kyasanur Forest Virus, West        Nile encephalitis virus, St. Louis encephalitis virus, Russian        spring-summer encephalitis virus, Powassan encephalitis virus.    -   Pestivirus: Viral immunogens include, but are not limited to,        those derived from a Pestivirus, such as Bovine viral diarrhea        (BVDV), Classical swine fever (CSFV) or Border disease (BDV).    -   Hepadnavirus: Viral immunogens include, but are not limited to,        those derived from a Hepadnavirus, such as Hepatitis B virus. A        composition can include hepatitis B virus surface antigen        (HBsAg).    -   Other hepatitis viruses: A composition can include an immunogen        from a hepatitis C virus, delta hepatitis virus, hepatitis E        virus, or hepatitis G virus.    -   Rhabdovirus: Viral immunogens include, but are not limited to,        those derived from a Rhabdovirus, such as a Lyssavirus (e.g. a        Rabies virus) and Vesiculovirus (VSV).    -   Caliciviridae: Viral immunogens include, but are not limited to,        those derived from Calciviridae, such as Norwalk virus        (Norovirus), and Norwalk-like Viruses, such as Hawaii Virus and        Snow Mountain Virus.    -   Coronavirus: Viral immunogens include, but are not limited to,        those derived from a SARS coronavirus, avian infectious        bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine        transmissible gastroenteritis virus (TGEV). The coronavirus        immunogen may be a spike polypeptide.    -   Retrovirus: Viral immunogens include, but are not limited to,        those derived from an Oncovirus, a Lentivirus (e.g. HIV-1 or        HIV-2) or a Spumavirus.    -   Reovirus: Viral immunogens include, but are not limited to,        those derived from an Orthoreovirus, a Rotavirus, an Orbivirus,        or a Coltivirus.    -   Parvovirus: Viral immunogens include, but are not limited to,        those derived from Parvovirus B19.    -   Herpesvirus: Viral immunogens include, but are not limited to,        those derived from a human herpesvirus, such as, by way of        example only, Herpes Simplex Viruses (HSV) (e.g. HSV types 1 and        2), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV),        Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human        Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8).    -   Papovaviruses: Viral immunogens include, but are not limited to,        those derived from Papillomaviruses and Polyomaviruses. The        (human) papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11,        13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65        e.g. from one or more of serotypes 6, 11, 16 and/or 18.    -   Adenovirus: Viral immunogens include those derived from        adenovirus serotype 36 (Ad-36).

In some embodiments, the immunogen elicits an immune response against avirus which infects fish, such as: infectious salmon anemia virus(ISAV), salmon pancreatic disease virus (SPDV), infectious pancreaticnecrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystisdisease virus (FLDV), infectious hematopoietic necrosis virus (IHNV),koi herpesvirus, salmon picorna-like virus (also known as picorna-likevirus of atlantic salmon), landlocked salmon virus (LSV), atlanticsalmon rotavirus (ASR), trout strawberry disease virus (TSD), cohosalmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).

Fungal immunogens may be derived from Dermatophytres, including:Epidermophyton floccusum, Microsporum audouini, Microsporum canis,Microsporum distortum, Microsporum equinum, Microsporum gypsum,Microsporum nanum, Trichophyton concentricum, Trichophyton equinum,Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini,Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophytonrubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophytonverrucosum, T verrucosum var. album, var. discoides, var. ochraceum,Trichophyton violaceum, and/or Trichophyton faviforme; or fromAspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi,Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus,Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata,Candida krusei, Candida parapsilosis, Candida stellatoidea, Candidakusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis,Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis,Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum,Histoplasma capsulatum, Klebsiella pneumoniae, Microsporidia,Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi;the less common are Brachiola spp, Microsporidium spp., Nosema spp.,Pleistophora spp., Trachipleistophora spp., Vittaforma sppParacoccidioides brasiliensis, Pneumocystis carinii, Pythiumninsidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomycesboulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrixschenckii, Trichosporon beigelii, Toxoplasma gondii, Penicilliummarneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrixspp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp,Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp.,Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp,Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp,Paecilomyces spp, Pithomyces spp, and Cladosporium spp.

In some embodiments the immunogen elicits an immune response against aparasite from the Plasmodium genus, such as P. falciparum, P. vivax, P.malariae or P. ovale. Thus the invention may be used for immunisingagainst malaria. In some embodiments the immunogen elicits an immuneresponse against a parasite from the Caligidae family, particularlythose from the Lepeophtheirus and Caligus genera e.g. sea lice such asLepeophtheirus salmonis or Caligus rogercresseyi.

In some embodiments the immunogen elicits an immune response against:pollen allergens (tree-, herb, weed-, and grass pollen allergens);insect or arachnid allergens (inhalant, saliva and venom allergens, e.g.mite allergens, cockroach and midges allergens, hymenopthera venomallergens); animal hair and dandruff allergens (from e.g. dog, cat,horse, rat, mouse, etc.); and food allergens (e.g. a gliadin). Importantpollen allergens from trees, grasses and herbs are such originating fromthe taxonomic orders of Fagales, Oleales, Pinales and platanaceaeincluding, but not limited to, birch (Betula), alder (Alnus), hazel(Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria andJuniperus), plane tree (Platanus), the order of Poales including grassesof the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris,Secale, and Sorghum, the orders of Asterales and Urticales includingherbs of the genera Ambrosia, Artemisia, and Parietaria. Other importantinhalation allergens are those from house dust mites of the genusDermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys,Glycyphagus and Tyrophagus, those from cockroaches, midges and flease.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and thosefrom mammals such as cat, dog and horse, venom allergens including suchoriginating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (Apidae), wasps(Vespidea), and ants (Formicoidae).

In some embodiments the immunogen is a tumor antigen selected from: (a)cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE,BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2,MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which canbe used, for example, to address melanoma, lung, head and neck, NSCLC,breast, gastrointestinal, and bladder tumors; (b) mutated antigens, forexample, p53 (associated with various solid tumors, e.g., colorectal,lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma,pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g.,melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associatedwith, e.g., head and neck cancer), CIA 0205 (associated with, e.g.,bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g.,melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma),BCR-abl (associated with, e.g., chronic myelogenous leukemia),triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c)over-expressed antigens, for example, Galectin 4 (associated with, e.g.,colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin'sdisease), proteinase 3 (associated with, e.g., chronic myelogenousleukemia), WT 1 (associated with, e.g., various leukemias), carbonicanhydrase (associated with, e.g., renal cancer), aldolase A (associatedwith, e.g., lung cancer), PRAME (associated with, e.g., melanoma),HER-2/neu (associated with, e.g., breast, colon, lung and ovariancancer), mammaglobin, alpha-fetoprotein (associated with, e.g.,hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin(associated with, e.g., pancreatic and gastric cancer), telomerasecatalytic protein, MUC-1 (associated with, e.g., breast and ovariancancer), G-250 (associated with, e.g., renal cell carcinoma), p53(associated with, e.g., breast, colon cancer), and carcinoembryonicantigen (associated with, e.g., breast cancer, lung cancer, and cancersof the gastrointestinal tract such as colorectal cancer); (d) sharedantigens, for example, melanoma-melanocyte differentiation antigens suchas MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma); (e) prostateassociated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer; (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example). In certainembodiments, tumor immunogens include, but are not limited to, p15,Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virusantigens, EBNA, human papillomavirus (HPV) antigens, including E6 andE7, hepatitis B and C virus antigens, human T-cell lymphotropic virusantigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4,791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM),HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16,TA-90 (Mac-2 binding protein/cyclophilin C-associated protein), TAAL6,TAG72, TLP, TPS, and the like.

Pharmaceutical Compositions

Particles of the invention are useful as components in pharmaceuticalcompositions for immunising subjects against various diseases. Thesecompositions will typically include a pharmaceutically acceptablecarrier in addition to the particles. A thorough discussion ofpharmaceutically acceptable carriers is available in reference 35.

A pharmaceutical composition of the invention may include one or moresmall molecule immunopotentiators. For example, the composition mayinclude a TLR2 agonist (e.g. Pam3CSK4), a TLR4 agonist (e.g. anaminoalkyl glucosaminide phosphate, such as E6020), a TLR7 agonist (e.g.imiquimod), a TLR8 agonist (e.g. resiquimod) and/or a TLR9 agonist (e.g.IC31). Any such agonist ideally has a molecular weight of <2000 Da.Where a RNA is encapsulated, in some embodiments such agonist(s) arealso encapsulated with the RNA, but in other embodiments they areunencapsulated. Where a RNA is adsorbed to a particle, in someembodiments such agonist(s) are also adsorbed with the RNA, but in otherembodiments they are unadsorbed.

Pharmaceutical compositions of the invention may include the particlesin plain water (e.g. w.f.i.) or in a buffer e.g. a phosphate buffer, aTris buffer, a borate buffer, a succinate buffer, a histidine buffer, ora citrate buffer. Buffer salts will typically be included in the 5-20 mMrange.

Pharmaceutical compositions of the invention may have a pH between 5.0and 9.5 e.g. between 6.0 and 8.0.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical e.g. about 9 mg/ml.

Compositions of the invention may include metal ion chelators. These canprolong RNA stability by removing ions which can acceleratephosphodiester hydrolysis. Thus a composition may include one or more ofEDTA, EGTA, BAPTA, pentetic acid, etc. Such chelators are typicallypresent at between 10-500 μM e.g. 0.1 mM. A citrate salt, such as sodiumcitrate, can also act as a chelator, while advantageously also providingbuffering activity.

Pharmaceutical compositions of the invention may have an osmolality ofbetween 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, orbetween 290-310 mOsm/kg.

Pharmaceutical compositions of the invention may include one or morepreservatives, such as thiomersal or 2-phenoxyethanol. Mercury-freecompositions are preferred, and preservative-free vaccines can beprepared.

Pharmaceutical compositions of the invention are preferably sterile.

Pharmaceutical compositions of the invention are preferablynon-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose.

Pharmaceutical compositions of the invention are preferably gluten free.

Pharmaceutical compositions of the invention may be prepared in unitdose form. In some embodiments a unit dose may have a volume of between0.1-1.0 ml e.g. about 0.5 m1.

The compositions may be prepared as injectables, either as solutions orsuspensions. The composition may be prepared for pulmonaryadministration e.g. by an inhaler, using a fine spray. The compositionmay be prepared for nasal, aural or ocular administration e.g. as sprayor drops. Injectables for intramuscular administration are typical.

Compositions comprise an immunologically effective amount of particles,as well as any other components, as needed. By ‘immunologicallyeffective amount’, it is meant that the administration of that amount toan individual, either in a single dose or as part of a series, iseffective for treatment or prevention. This amount varies depending uponthe health and physical condition of the individual to be treated, age,the taxonomic group of individual to be treated (e.g. non-human primate,primate, etc.), the capacity of the individual's immune system tosynthesise antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials. The particle and RNA content of compositions of theinvention will generally be expressed in terms of the amount of RNA perdose. A preferred dose has ≤100 μg RNA (e.g. from 10-100 μg, such asabout 10 μg, 25 μg, 50 μg, 75 μg or 100 μg), but expression can be seenat much lower levels e.g. ≤1 μg/dose, ≤100 μg/dose, ≤10 μg/dose, ≤1μg/dose, etc

The invention also provides a delivery device (e.g. syringe, nebuliser,sprayer, inhaler, dermal patch, etc.) containing a pharmaceuticalcomposition of the invention. This device can be used to administer thecomposition to a vertebrate subject.

Particles of the invention do not include ribosomes.

Methods of Treatment and Medical Uses

In contrast to the particles disclosed in reference 16, particles andpharmaceutical compositions of the invention are for in vivo use foreliciting an immune response against an immunogen of interest.

The invention provides a method for raising an immune response in avertebrate comprising the step of administering an effective amount of aparticle or pharmaceutical composition of the invention. The immuneresponse is preferably protective and preferably involves antibodiesand/or cell-mediated immunity. The method may raise a booster response.

The invention also provides a particle or pharmaceutical composition ofthe invention for use in a method for raising an immune response in avertebrate.

The invention also provides the use of a particle of the invention inthe manufacture of a medicament for raising an immune response in avertebrate.

By raising an immune response in the vertebrate by these uses andmethods, the vertebrate can be protected against various diseases and/orinfections e.g. against bacterial and/or viral diseases as discussedabove. The particles and compositions are immunogenic, and are morepreferably vaccine compositions. Vaccines according to the invention mayeither be prophylactic (i.e. to prevent infection) or therapeutic (i.e.to treat infection), but will typically be prophylactic.

The vertebrate is preferably a mammal, such as a human or a largeveterinary mammal (e.g. horses, cattle, deer, goats, pigs). Where thevaccine is for prophylactic use, the human is preferably a child (e.g. atoddler or infant) or a teenager; where the vaccine is for therapeuticuse, the human is preferably a teenager or an adult. A vaccine intendedfor children may also be administered to adults e.g. to assess safety,dosage, immunogenicity, etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Thus a human patient may be less than 1 year old,less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old,or at least 55 years old. Preferred patients for receiving the vaccinesare the elderly (e.g. ≥50 years old, ≥60 years old, and preferably ≥65years), the young (e.g. ≤5 years old), hospitalised patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, or immunodeficient patients. The vaccines are notsuitable solely for these groups, however, and may be used moregenerally in a population.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,intradermally, or to the interstitial space of a tissue; unlikereference 1, intraglossal injection is not typically used with thepresent invention). Alternative delivery routes include rectal, oral(e.g. tablet, spray), buccal, sublingual, vaginal, topical, transdermalor transcutaneous, intranasal, ocular, aural, pulmonary or other mucosaladministration. Intradermal and intramuscular administration are twopreferred routes. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose is 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity,preferably to elicit an enhanced systemic and/or mucosal immunity.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In oneembodiment, multiple doses may be administered approximately 6 weeks, 10weeks and 14 weeks after birth, e.g. at an age of 6 weeks, 10 weeks and14 weeks, as often used in the World Health Organisation's ExpandedProgram on Immunisation (“EPI”). In an alternative embodiment, twoprimary doses are administered about two months apart, e.g. about 7, 8or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the second primary dose, e.g. about 6, 8, 10 or 12months after the second primary dose. In a further embodiment, threeprimary doses are administered about two months apart, e.g. about 7, 8or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the third primary dose, e.g. about 6, 8, 10, or 12months after the third primary dose.

GENERAL EMBODIMENTS

In some embodiments of the invention, the RNA includes no modifiednucleotides (see above). In other embodiments the RNA can optionallyinclude at least one modified nucleotide, provided that one or more ofthe following features (already disclosed above) is also required:

-   -   A. Where the RNA is delivered with a liposome, the liposome        comprises DSDMA, DODMA, DLinDMA and/or DLenDMA.    -   B. Where the RNA is encapsulated in a liposome, the hydrophilic        portion of a lipid in the liposome is PEGylated.    -   C. Where the RNA is encapsulated in a liposome, at least 80% by        number of the liposomes have diameters in the range of 20-220        nm.    -   D. Where the RNA is delivered with a microparticle, the        microparticle is a non-toxic and biodegradable polymer        microparticle.    -   E. Where the RNA is delivered with a microparticle, the        microparticles have a diameter in the range of 0.02 μm to 8 μm.    -   F. Where the RNA is delivered with a microparticle, at least 80%        by number of the microparticles have a diameter in the range of        0.03-7 μm.    -   G. Where the RNA is delivered with a microparticle, the        composition is lyophilised.    -   H. The RNA has a 3′ poly-A tail, and the immunogen can elicits        an immune response in vivo against a bacterium, a virus, a        fungus or a parasite.    -   I. The RNA is delivered in combination with a metal ion chelator        with a delivery system selected from (i) liposomes (ii)        non-toxic and biodegradable polymer microparticles.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references36-42, etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to charge, to cations, to anions, to zwitterions, etc., aretaken at pH 7.

TLR3 is the Toll-like receptor 3. It is a single membrane-spanningreceptor which plays a key role in the innate immune system. Known TLR3agonists include poly(I:C). “TLR3” is the approved HGNC name for thegene encoding this receptor, and its unique HGNC ID is HGNC:11849. TheRefSeq sequence for the human TLR3 gene is GI:2459625.

TLR7 is the Toll-like receptor 7. It is a single membrane-spanningreceptor which plays a key role in the innate immune system. Known TLR7agonists include e.g. imiquimod. “TLR7” is the approved HGNC name forthe gene encoding this receptor, and its unique HGNC ID is HGNC:15631.The RefSeq sequence for the human TLR7 gene is GI:67944638.

TLR8 is the Toll-like receptor 8. It is a single membrane-spanningreceptor which plays a key role in the innate immune system. Known TLR8agonists include e.g. resiquimod. “TLR8” is the approved HGNC name forthe gene encoding this receptor, and its unique HGNC ID is HGNC:15632.The RefSeq sequence for the human TLR8 gene is GI:20302165.

The RIG-I-like receptor (“RLR”) family includes various RNA helicaseswhich play key roles in the innate immune system[43]. RLR-1 (also knownas RIG-I or retinoic acid inducible gene I) has two caspase recruitmentdomains near its N-terminus. The approved HGNC name for the geneencoding the RLR-1 helicase is “DDX58” (for DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 58) and the unique HGNC ID is HGNC:19102. The RefSeqsequence for the human RLR-1 gene is GI:77732514. RLR-2 (also known asMDA5 or melanoma differentiation-associated gene 5) also has two caspaserecruitment domains near its N-terminus. The approved HGNC name for thegene encoding the RLR-2 helicase is “IFIH1” (for interferon induced withhelicase C domain 1) and the unique HGNC ID is HGNC:18873. The RefSeqsequence for the human RLR-2 gene is GI: 27886567. RLR-3 (also known asLGP2 or laboratory of genetics and physiology 2) has no caspaserecruitment domains. The approved HGNC name for the gene encoding theRLR-3 helicase is “DHX58” (for DEXH (Asp-Glu-X-His) box polypeptide 58)and the unique HGNC ID is HGNC:29517. The RefSeq sequence for the humanRLR-3 gene is GI:149408121.

PKR is a double-stranded RNA-dependent protein kinase. It plays a keyrole in the innate immune system. “EIF2AK2” (for eukaryotic translationinitiation factor 2-alpha kinase 2) is the approved HGNC name for thegene encoding this enzyme, and its unique HGNC ID is HGNC:9437. TheRefSeq sequence for the human PKR gene is GI:208431825.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a gel with stained RNA. Lanes show (1) markers (2) nakedreplicon (3) replicon after RNase treatment (4) replicon encapsulated inliposome (5) liposome after RNase treatment (6) liposome treated withRNase then subjected to phenol/chloroform extraction.

FIG. 2 is an electron micrograph of liposomes.

FIG. 3 shows protein expression (as relative light units, RLU) at days1, 3 and 6 after delivery of RNA as a virion-packaged replicon(squares), naked RNA (triangles), or as microparticles (circles).

FIG. 4 shows a gel with stained RNA. Lanes show (1) markers (2) nakedreplicon (3) replicon encapsulated in liposome (4) liposome treated withRNase then subjected to phenol/chloroform extraction.

FIG. 5 shows protein expression at days 1, 3 and 6 after delivery of RNAas a virion-packaged replicon (squares), as naked RNA (diamonds), or inliposomes (+=0.1 μg, x=1 μg).

FIG. 6 shows protein expression at days 1, 3 and 6 after delivery offour different doses of liposome-encapsulated RNA.

FIG. 7 shows anti-F IgG titers in animals receiving virion-packagedreplicon (VRP or VSRP), 1 μg naked RNA, and 1 μg liposome-encapsulatedRNA.

FIG. 8 shows anti-F IgG titers in animals receiving VRP, 1 μg naked RNA,and 0.1 g or 1 μg liposome-encapsulated RNA.

FIG. 9 shows neutralising antibody titers in animals receiving VRP oreither 0.1 g or 1 μg liposome-encapsulated RNA.

FIG. 10 shows expression levels after delivery of a replicon as nakedRNA (circles), liposome-encapsulated RNA (triangle & square), or as alipoplex (inverted triangle).

FIG. 11 shows F-specific IgG titers (2 weeks after second dose) afterdelivery of a replicon as naked RNA (0.01-1 μg), liposome-encapsulatedRNA (0.01-10 μg), or packaged as a virion (VRP, 10⁶ infectious units orIU).

FIG. 12 shows F-specific IgG titers (circles) and PRNT titers (squares)after delivery of a replicon as naked RNA (1 μg), liposome-encapsulatedRNA (0.1 or 1 μg), or packaged as a virion (VRP, 10⁶ IU). Titers innaïve mice are also shown. Solid lines show geometric means.

FIG. 13 shows intracellular cytokine production after restimulation withsynthetic peptides representing the major epitopes in the F protein, 4weeks after a second dose. The y-axis shows the % cytokine+ of CD8+CD4−.

FIG. 14 shows F-specific IgG titers (mean log₁₀ titers±std dev) over 63days (FIG. 14A) and 210 days (FIG. 14B) after immunisation of calves.The three lines are easily distinguished at day 63 and are, from bottomto top: PBS negative control; liposome-delivered RNA; and the “Triangle4” product.

FIG. 15 shows anti-HIV serum IgG titers in response to naked (“RNA”) orliposome-encapsulated (“LNP”) RNA, or to DNA delivered by electroporatedinto muscle.

FIG. 16 shows IgG titers in 13 groups of mice. Each circle is anindividual mouse, and solid lines show geometric means. The dottedhorizontal line is the assay's detection limit. The 13 groups are, fromleft to right, A to M as described below.

FIG. 17 shows (A) IL-6 and (B) IFNα (pg/ml) released by pDC. There are 4pairs of bars, from left to right: control; immunised with RNA+DOTAP;immunised with RNA+lipofectamine; and immunised with RNA in liposomes.In each pair the black bar is wild-type mice, grey is rsq1 mutant.

MODES FOR CARRYING OUT THE INVENTION

RNA Replicons

Various replicons are used below. In general these are based on a hybridalphavirus genome with non-structural proteins from venezuelan equineencephalitis virus (VEEV), a packaging signal from sindbis virus, and a3′ UTR from Sindbis virus or a VEEV mutant. The replicon is about 10 kblong and has a poly-A tail.

Plasmid DNA encoding alphavirus replicons (named: pT7-mVEEV-FL.RSVF orA317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) served as a templatefor synthesis of RNA in vitro. The replicons contain the alphavirusgenetic elements required for RNA replication but lack those encodinggene products necessary for particle assembly; the structural proteinsare instead replaced by a protein of interest (either a reporter, suchas SEAP or GFP, or an immunogen, such as full-length RSV F protein) andso the replicons are incapable of inducing the generation of infectiousparticles. A bacteriophage (T7 or SP6) promoter upstream of thealphavirus cDNA facilitates the synthesis of the replicon RNA in vitroand a hepatitis delta virus (HDV) ribozyme immediately downstream of thepoly(A)-tail generates the correct 3′-end through its self-cleavingactivity.

Following linearization of the plasmid DNA downstream of the HDVribozyme with a suitable restriction endonuclease, run-off transcriptswere synthesized in vitro using T7 or SP6 bacteriophage derivedDNA-dependent RNA polymerase. Transcriptions were performed for 2 hoursat 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNApolymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP andUTP) following the instructions provided by the manufacturer (Ambion).Following transcription the template DNA was digested with TURBO DNase(Ambion). The replicon RNA was precipitated with LiCl and reconstitutedin nuclease-free water. Uncapped RNA was capped post-transcriptionallywith Vaccinia Capping Enzyme (VCE) using the ScriptCap m7G CappingSystem (Epicentre Biotechnologies) as outlined in the user manual;replicons capped in this way are given the “v” prefix e.g. vA317 is theA317 replicon capped by VCE. Post-transcriptionally capped RNA wasprecipitated with LiCl and reconstituted in nuclease-free water. Theconcentration of the RNA samples was determined by measuring OD_(260nm).Integrity of the in vitro transcripts was confirmed by denaturingagarose gel electrophoresis.

PLG Adsorption

Microparticles were made using 500 mg of PLG RG503 (50:50lactide/glycolide molar ratio, MW ˜30 kDa) and 20 mg DOTAP using an OmniMacro Homogenizer. The particle suspension was shaken at 150 rpmovernight and then filtered through a 40 μm sterile filter for storageat 2-8° C. Self-replicating RNA was adsorbed to the particles. Toprepare 1 mL of PLG/RNA suspension the required volume of PLG particlesuspension was added to a vial and nuclease-free water was added tobring the volume to 900 μL. 100 μL RNA (10 μg/mL) was added dropwise tothe PLG suspension, with constant shaking. PLG/RNA was incubated at roomtemperature for 30 min. For 1 mL of reconstituted suspension, 45 mgmannitol, 15 mg sucrose and 250-500 μg of PVA were added. The vials werefrozen at −80° C. and lyophilized.

To evaluate RNA adsorption, 100 μL particle suspension was centrifugedat 10,000 rpm for 5 min and supernatant was collected. PLG/RNA wasreconstituted using 1 mL nuclease-free water. To 100 μL particlesuspension (1 μg RNA), 1 mg heparin sulfate was added. The mixture wasvortexed and allowed to sit at room temperature for 30 min for RNAdesorption. Particle suspension was centrifuged and supernatant wascollected.

For RNAse stability, 100 μL particle suspension was incubated with 6.4mAU of RNase A at room temperature for 30 min. RNAse was inactivatedwith 0.126 mAU of Proteinase K at 55° C. for 10 min. 1 mg of heparinsulfate was added to desorb the RNA followed by centrifugation. Thesupernatant samples containing RNA were mixed with formaldehyde loaddye, heated at 65° C. for 10 min and analyzed using a 1% denaturing gel(460 ng RNA loaded per lane).

To assess expression, Balb/c mice were immunized with 1 μg RNA in 100 μLintramuscular injection volume (50 μL/leg) on day 0. Sera were collectedon days 1, 3 and 6. Protein expression was determined using achemiluminescence assay. As shown in FIG. 3, expression was higher whenRNA was delivered by PLG (triangles) than without any delivery particle(circles).

Liposomal Encapsulation

RNA was encapsulated in liposomes made by the method of references 7 and44. The liposomes were made of 10% DSPC (zwitterionic), 40% DlinDMA(cationic), 48% cholesterol and 2% PEG-conjugated DMG (2 kDa PEG). Theseproportions refer to the % moles in the total liposome.

DlinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesizedusing the procedure of reference 2. DSPC(1,2-Diastearoyl-sn-glycero-3-phosphocholine) was purchased fromGenzyme. Cholesterol was obtained from Sigma-Aldrich. PEG-conjugated DMG(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol), ammonium salt), DOTAP(1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and DC-chol(3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride)were from Avanti Polar Lipids.

Briefly, lipids were dissolved in ethanol (2 ml), a RNA replicon wasdissolved in buffer (2 ml, 100 mM sodium citrate, pH 6) and these weremixed with 2 ml of buffer followed by 1 hour of equilibration. Themixture was diluted with 6 ml buffer then filtered. The resultingproduct contained liposomes, with ˜95% encapsulation efficiency.

For example, in one particular method, fresh lipid stock solutions wereprepared in ethanol. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg ofcholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 mLof ethanol. The freshly prepared lipid stock solution was gently rockedat 37° C. for about 15 min to form a homogenous mixture. Then, 755 μL ofthe stock was added to 1.245 mL ethanol to make a working lipid stocksolution of 2 mL. This amount of lipids was used to form liposomes with250 μg RNA. A 2 mL working solution of RNA was also prepared from astock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mLglass vials (with stir bars) were rinsed with RNase Away solution(Molecular BioProducts) and washed with plenty of MilliQ water beforeuse to decontaminate the vials of RNases. One of the vials was used forthe RNA working solution and the others for collecting the lipid and RNAmixes (as described later). The working lipid and RNA solutions wereheated at 37° C. for 10 min before being loaded into 3 cc luer-loksyringes. 2 mL citrate buffer (pH 6) was loaded in another 3 cc syringe.Syringes containing RNA and the lipids were connected to a T mixer(PEEK™ 500 μm ID junction, Idex Health Science) using FEP tubing(fluorinated ethylene-propylene; all FEP tubing used had a 2 mm internaldiameter and a 3 mm outer diameter; obtained from Idex Health Science).The outlet from the T mixer was also FEP tubing. The third syringecontaining the citrate buffer was connected to a separate piece oftubing. All syringes were then driven at a flow rate of 7 mL/min using asyringe pump. The tube outlets were positioned to collect the mixturesin a 20 mL glass vial (while stirring). The stir bar was taken out andthe ethanol/aqueous solution was allowed to equilibrate to roomtemperature for 1 h. 4 ml of the mixture was loaded into a 5 cc syringe,which was connected to a piece of FEP tubing and in another 5 cc syringeconnected to an equal length of FEP tubing, an equal amount of 100 mMcitrate buffer (pH 6) was loaded. The two syringes were driven at 7mL/min flow rate using the syringe pump and the final mixture collectedin a 20 mL glass vial (while stirring). Next, the mixture collected fromthe second mixing step (liposomes) were passed through a Mustang Qmembrane (an anion-exchange support that binds and removes anionicmolecules, obtained from Pall Corporation). Before using this membranefor the liposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100mM citrate buffer (pH 6) were successively passed through it. Liposomeswere warmed for 10 min at 37° C. before passing through the membrane.Next, liposomes were concentrated to 2 mL and dialyzed against 10-15volumes of 1×PBS using by tangential flow filtration before recoveringthe final product. The TFF system and hollow fiber filtration membraneswere purchased from Spectrum Labs (Rancho Dominguez) and were usedaccording to the manufacturer's guidelines. Polysulfone hollow fiberfiltration membranes with a 100 kD pore size cutoff and 8 cm² surfacearea were used. For in vitro and in vivo experiments formulations werediluted to the required RNA concentration with 1×PBS. Further liposomemanufacturing methods are disclosed below.

FIG. 2 shows an example electron micrograph of liposomes prepared bythese methods. These liposomes contain encapsulated RNA encodingfull-length RSV F antigen. Dynamic light scattering of one batch showedan average diameter of 141 nm (by intensity) or 78 nm (by number).

The percentage of encapsulated RNA and RNA concentration were determinedby Quant-iT RiboGreen RNA reagent kit (Invitrogen), followingmanufacturer's instructions. The ribosomal RNA standard provided in thekit was used to generate a standard curve. Liposomes were diluted 10× or100× in 1×TE buffer (from kit) before addition of the dye. Separately,liposomes were diluted 10× or 100× in 1×TE buffer containing 0.5% TritonX before addition of the dye (to disrupt the liposomes and thus to assaytotal RNA). Thereafter an equal amount of dye was added to each solutionand then ˜180 μL of each solution after dye addition was loaded induplicate into a 96 well tissue culture plate. The fluorescence (Ex 485nm, Em 528 nm) was read on a microplate reader. All liposomeformulations were dosed in vivo based on the encapsulated amount of RNA.

Encapsulation in liposomes was shown to protect RNA from RNasedigestion. Experiments used 3.8 mAU of RNase A per microgram of RNA,incubated for 30 minutes at room temperature. RNase was inactivated withProteinase K at 55° C. for 10 minutes. A 1:1 v/v mixture of sample to25:24:1 v/v/v, phenol:chloroform:isoamyl alcohol was then added toextract the RNA from the lipids into the aqueous phase. Samples weremixed by vortexing for a few seconds and then placed on a centrifuge for15 minutes at 12 k RPM. The aqueous phase (containing the RNA) wasremoved and used to analyze the RNA. Prior to loading (400 ng RNA perwell) all the samples were incubated with formaldehyde loading dye,denatured for 10 minutes at 65° C. and cooled to room temperature.Ambion Millennium markers were used to approximate the molecular weightof the RNA construct. The gel was run at 90 V. The gel was stained using0.1% SYBR gold according to the manufacturer's guidelines in water byrocking at room temperature for 1 hour. FIG. 1 shows that RNasecompletely digests RNA in the absence of encapsulation (lane 3). RNA isundetectable after encapsulation (lane 4), and no change is seen ifthese liposomes are treated with RNase (lane 4). After RNase-treatedliposomes are subjected to phenol extraction, undigested RNA is seen(lane 6).

Even after 1 week at 4° C. the RNA could be seen without anyfragmentation (FIG. 4, arrow). Protein expression in vivo was unchangedafter 6 weeks at 4° C. and one freeze-thaw cycle. Thusliposome-encapsulated RNA is stable.

To assess in vivo expression of the RNA a reporter enzyme (SEAP;secreted alkaline phosphatase) was encoded in the replicon, rather thanan immunogen. Expression levels were measured in sera diluted 1:4 in 1×Phospha-Light dilution buffer using a chemiluminescent alkalinephosphate substrate. 8-10 week old BALB/c mice (5/group) were injectedintramuscularly on day 0, 50 μl per leg with 0.1 μg or 1 μg RNA dose.The same vector was also administered without the liposomes (in RNasefree 1×PBS) at 1 μg. Virion-packaged replicons were also tested.Virion-packaged replicons used herein (referred to as “VRPs”) wereobtained by the methods of reference 45, where the alphavirus repliconis derived from the mutant VEEV or a chimera derived from the genome ofVEEV engineered to contain the 3′ UTR of Sindbis virus and a Sindbisvirus packaging signal (PS), packaged by co-electroporating them intoBHK cells with defective helper RNAs encoding the Sindbis virus capsidand glycoprotein genes.

As shown in FIG. 5, encapsulation increased SEAP levels by about ½ logat the 1 μg dose, and at day 6 expression from a 0.1 μg encapsulateddose matched levels seen with 1 μg unencapsulated dose. By day 3expression levels exceeded those achieved with VRPs (squares). Thusexpressed increased when the RNA was formulated in the liposomesrelative to the naked RNA control, even at a 10× lower dose. Expressionwas also higher relative to the VRP control, but the kinetics ofexpression were very different (see FIG. 5). Delivery of the RNA withelectroporation resulted in increased expression relative to the nakedRNA control, but these levels were lower than with liposomes.

Further SEAP experiments showed a clear dose response in vivo, withexpression seen after delivery of as little as 1 ng RNA (FIG. 6).Further experiments comparing expression from encapsulated and nakedreplicons indicated that 0.01 μg encapsulated RNA was equivalent to 1 μgof naked RNA. At a 0.5 μg dose of RNA the encapsulated material gave a12-fold higher expression at day 6; at a 0.1 μg dose levels were 24-foldhigher at day 6.

Rather than looking at average levels in the group, individual animalswere also studied. Whereas several animals were non-responders to nakedreplicons, encapsulation eliminated non-responders.

Further experiments replaced DlinDMA with DOTAP. Although the DOTAPliposomes gave better expression than naked replicon, they were inferiorto the DlinDMA liposomes (2- to 3-fold difference at day 1).

To assess in vivo immunogenicity a replicon was constructed to expressfull-length F protein from respiratory syncytial virus (RSV). This wasdelivered naked (1 μg), encapsulated in liposomes (0.1 or 1 μg), orpackaged in virions (10⁶ IU; “VRP”) at days 0 and 21. FIG. 7 showsanti-F IgG titers 2 weeks after the second dose, and the liposomesclearly enhance immunogenicity. FIG. 8 shows titers 2 weeks later, bywhich point there was no statistical difference between the encapsulatedRNA at 0.1 μg, the encapsulated RNA at 1 μg, or the VRP group.Neutralisation titers (measured as 60% plaque reduction, “PRNT60”) werenot significantly different in these three groups 2 weeks after thesecond dose (FIG. 9). FIG. 12 shows both IgG and PRNT titers 4 weeksafter the second dose.

FIG. 13 confirms that the RNA elicits a robust CD8 T cell response.

Further experiments compared F-specific IgG titers in mice receivingVRP, 0.1 μg liposome-encapsulated RNA, or 1 μg liposome-encapsulatedRNA. Titer ratios (VRP: liposome) at various times after the second dosewere as follows:

2 weeks 4 weeks 8 weeks 0.1 μg   2.9 1.0 1.1 1 μg 2.3 0.9 0.9

Thus the liposome-encapsulated RNA induces essentially the samemagnitude of immune response as seen with virion delivery.

Further experiments showed superior F-specific IgG responses with a 10μg dose, equivalent responses for 1 μg and 0.1 μg doses, and a lowerresponse with a 0.01 μg dose. FIG. 11 shows IgG titers in mice receivingthe replicon in naked form at 3 different doses, in liposomes at 4different doses, or as VRP (10⁶ IU). The response seen with 1 μgliposome-encapsulated RNA was statistically insignificant (ANOVA) whencompared to VRP, but the higher response seen with 10 μgliposome-encapsulated RNA was statistically significant (p<0.05) whencompared to both of these groups.

A further study confirmed that the 0.1 μg of liposome-encapsulated RNAgave much higher anti-F IgG responses (15 days post-second dose) than0.1 μg of delivered DNA, and even was more immunogenic than 20 μgplasmid DNA encoding the F antigen, delivered by electroporation (Elgen™DNA Delivery System, Inovio).

Mice showed few visual signs of distress (weight loss, etc.) afterreceiving liposome-encapsulated RNA replicon, although a transientweight loss of 3-4% was seen after a second dose of 10 μg RNA. Incontrast, delivery of 10 μg liposome-encapsulated DNA led to 8-10%weight loss.

Mechanism of Action

Bone marrow derived dendritic cells (pDC) were obtained from wild-typemice or the “Resq” (rsq1) mutant strain. The mutant strain has a pointmutation at the amino terminus of its TLR7 receptor which abolishes TLR7signalling without affecting ligand binding [46]. The cells werestimulated with replicon RNA formulated with DOTAP, lipofectamine 2000or inside a liposome. As shown in FIG. 17, IL-6 and INFα were induced inWT cells but this response was almost completely abrogated in mutantmice. These results shows that TLR7 is required for RNA recognition inimmune cells, and that liposome-encapsulated replicons can cause immunecells to secrete high levels of both interferons and pro-inflammatorycytokines.

In general, liposome-delivered RNA replicons were shown to induceseveral serum cytokines within 24 hours of intramuscular injection(IFN-α, IP-10 (CXCL-10), IL-6, KC, IL-5, IL-13, MCP-1, and MIP-a),whereas only MIP-1 was induced by naked RNA and liposome alone inducedonly IL-6.

IFN-α was shown to contribute to the immune response toliposome-encapsulated RSV-F-encoding replicon because an anti-IFNαreceptor (IFNAR1) antibody reduced F-specific serum IgG a 10-foldreduction after 2 vaccinations.

Liposome-delivered RNA replicons have generally been seen to elicit abalanced IgG1:IgG2a subtype profile in mice, sometimes with a higherIgG2a/IgG1 ratio than seen with electroporated DNA or with protein/MF59immunizations (i.e. a Th1-type immune response).

Liposome Manufacturing Methods

In general, eight different methods have been used for preparingliposomes according to the invention. These are referred to in the textas methods (A) to (H) and they differ mainly in relation to filtrationand TFF steps. Details are as follows:

-   -   (A) Fresh lipid stock solutions in ethanol were prepared. 37 mg        of DlinDMA, 11.8 mg of DSPC, 27.8 mg of Cholesterol and 8.07 mg        of PEG DMG 2000 were weighed and dissolved in 7.55 mL of        ethanol. The freshly prepared lipid stock solution was gently        rocked at 37° C. for about 15 min to form a homogenous mixture.        Then, 755 μL of the stock was added to 1.245 mL ethanol to make        a working lipid stock solution of 2 mL. This amount of lipids        was used to form liposomes with 250 μg RNA. A 2 mL working        solution of RNA was also prepared from a stock solution of ˜1        μg/μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials        (with stir bars) were rinsed with RNase Away solution (Molecular        BioProducts, San Diego, Calif.) and washed with plenty of MilliQ        water before use to decontaminate the vials of RNases. One of        the vials was used for the RNA working solution and the others        for collecting the lipid and RNA mixes (as described later). The        working lipid and RNA solutions were heated at 37° C. for 10 min        before being loaded into 3 cc luer-lok syringes. 2 mL of citrate        buffer (pH 6) was loaded in another 3 cc syringe. Syringes        containing RNA and the lipids were connected to a T mixer (PEEK™        500 μm ID junction, Idex Health Science, Oak Harbor, Wash.)        using FEP tubing (fluorinated ethylene-propylene; al FEP tubing        has a 2 mm internal diameter×3 mm outer diameter, supplied by        Idex Health Science). The outlet from the T mixer was also FEP        tubing. The third syringe containing the citrate buffer was        connected to a separate piece of FEP tubing. All syringes were        then driven at a flow rate of 7 mL/min using a syringe pump. The        tube outlets were positioned to collect the mixtures in a 20 mL        glass vial (while stirring). The stir bar was taken out and the        ethanol/aqueous solution was allowed to equilibrate to room        temperature for 1 hour. 4 ml of the mixture was loaded into a 5        cc syringe, which was connected to a piece of FEP tubing and in        another 5 cc syringe connected to an equal length of FEP tubing,        an equal amount of 100 mM citrate buffer (pH 6) was loaded. The        two syringes were driven at 7 mL/min flow rate using the syringe        pump and the final mixture collected in a 20 mL glass vial        (while stirring). Next, the mixture collected from the second        mixing step (liposomes) were passed through a Mustang Q membrane        (an anion-exchange support that binds and removes anionic        molecules, obtained from Pall Corporation, AnnArbor, Mich.,        USA). Before passing the liposomes, 4 mL of 1 M NaOH, 4 mL of 1        M NaCl and 10 mL of 100 mM citrate buffer (pH 6) were        successively passed through the Mustang membrane. Liposomes were        warmed for 10 min at 37° C. before passing through the membrane.        Next, liposomes were concentrated to 2 mL and dialyzed against        10-15 volumes of 1×PBS using TFF before recovering the final        product. The TFF system and hollow fiber filtration membranes        were purchased from Spectrum Labs and were used according to the        manufacturer's guidelines. Polysulfone hollow fiber filtration        membranes (part number P/N: X1AB-100-20P) with a 100 kD pore        size cutoff and 8 cm² surface area were used. For in vitro and        in vivo experiments, formulations were diluted to the required        RNA concentration with 1×PBS.    -   (B) As method (A) except that, after rocking, 226.7 μL of the        stock was added to 1.773 mL ethanol to make a working lipid        stock solution of 2 mL, thus modifying the lipid:RNA ratio.    -   (C) As method (B) except that the Mustang filtration was        omitted, so liposomes went from the 20 mL glass vial into the        TFF dialysis.    -   (D) As method (C) except that the TFF used polyethersulfone        (PES) hollow fiber membranes (part number P-C1-100E-100-01N)        with a 100 kD pore size cutoff and 20 cm² surface area.    -   (E) As method (D) except that a Mustang membrane was used, as in        method (A).    -   (F) As method (A) except that the Mustang filtration was        omitted, so liposomes went from the 20 mL glass vial into the        TFF dialysis.    -   (G) As method (D) except that a 4 mL working solution of RNA was        prepared from a stock solution of ˜1 μg/μL in 100 mM citrate        buffer (pH 6). Then four 20 mL glass vials were prepared in the        same way. Two of them were used for the RNA working solution (2        mL in each vial) and the others for collecting the lipid and RNA        mixes, as in (C). Rather than use T mixer, syringes containing        RNA and the lipids were connected to a Mitos Droplet junction        Chip (a glass microfluidic device obtained from Syrris, Part        no. 3000158) using PTFE tubing (0.03 inches internal diameter×        1/16 inch outer diameter) using a 4-way edge connector (Syrris).        Two RNA streams and one lipid stream were driven by syringe        pumps and the mixing of the ethanol and aqueous phase was done        at the X junction (100 μm×105 μm) of the chip. The flow rate of        all three streams was kept at 1.5 mL/min, hence the ratio of        total aqueous to ethanolic flow rate was 2:1. The tube outlet        was positioned to collect the mixtures in a 20 mL glass vial        (while stirring). The stir bar was taken out and the        ethanol/aqueous solution was allowed to equilibrate to room        temperature for 1 h. Then the mixture was loaded in a 5 cc        syringe, which was fitted to another piece of the PTFE tubing;        in another 5 cc syringe with equal length of PTFE tubing, an        equal volume of 100 mM citrate buffer (pH 6) was loaded. The two        syringes were driven at 3 mL/min flow rate using a syringe pump        and the final mixture collected in a 20 mL glass vial (while        stirring). Next, liposomes were concentrated to 2 mL and        dialyzed against 10-15 volumes of 1×PBS using TFF, as in (D).    -   (H) As method (A) except that the 2 mL working lipid stock        solution was made by mixing 120.9 μL of the lipid stock with        1.879 mL ethanol. Also, after mixing in the T mixer the        liposomes from the 20 mL vial were loaded into Pierce        Slide-A-Lyzer Dialysis Cassette (Thermo Scientific, extra        strength, 0.5-3 mL capacity) and dialyzed against 400-500 mL of        1×PBS overnight at 4° C. in an autoclaved plastic container        before recovering the final product.

BHK Expression

Liposomes with different lipids were incubated with BHK cells overnightand assessed for protein expression potency. From a baseline with RV05lipid expression could be increased 18× by adding 10%1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) to theliposome, 10× by adding 10% 18:2 (cis) phosphatidylcholine, and 900× byinstead using RV01.

In general, in vivo studies showed that unsaturated lipid tails tend toenhance IgG titers raised against encoded antigens.

RSV Immunogenicity

The vA317 self-replicating replicon encoding RSV F protein wasadministered to BALB/c mice, 4 or 8 animals per group, by bilateralintramuscular vaccinations (50 μL per leg) on days 0 and 21 with thereplicon (1 μg) alone or formulated as liposomes with DlinDMA (“RV01”)or DOTAP (“RV13”). The RV01 liposomes had 40% DlinDMA, 10% DSPC, 48%cholesterol and 2% PEG-DMG, but with differing amounts of RNA. The RV13liposomes had 40% DOTAP, 10% DPE, 48% cholesterol and 2% PEG-DMG. Forcomparison, naked plasmid DNA (20 μg) expressing the same RSV-F antigenwas delivered either using electroporation or with RV01(10) liposomes(0.1 μg DNA). Four mice were used as a naïve control group.

Liposomes were prepared by method (D) or method (B). For some liposomesmade by method (D) a double or half amount of RNA was used. The Zaverage particle diameter, polydispersity index and encapsulationefficiency of the liposomes were as follows:

RV Zav (nm) pdI % encapsulation Preparation RV01 (10) 158.6 0.088 90.7(A) RV01 (08) 156.8 0.144 88.6 (A) RV01 (05) 136.5 0.136 99   (B) RV01(09) 153.2 0.067 76.7 (A) RV01 (10) 134.7 0.147   87.8 * (A) RV13 (02)128.3 0.179 97   (A) * For this RV01(10) formulation the nucleic acidwas DNA not RNA

Serum was collected for antibody analysis on days 14, 36 and 49. Spleenswere harvested from mice at day 49 for T cell analysis.

F-specific serum IgG titers (GMT) were as follows:

RV Day 14 Day 36 Naked DNA plasmid 439 6712 Naked A317 RNA 78 2291 RV01(10) 3020 26170 RV01 (08) 2326 9720 RV01 (05) 5352 54907 RV01 (09) 442851316 RV01 (10) DNA 5 13 RV13 (02) 644 3616

The proportion of T cells which are cytokine-positive and specific forRSV F51-66 peptide are as follows, showing only figures which arestatistically significantly above zero:

CD4+CD8− CD4−CD8+ RV IFNγ IL2 IL5 TNFα IFNγ IL2 IL5 TNFα Naked 0.04 0.070.10 0.57 0.29 0.66 DNA plasmid Naked 0.04 0.05 0.08 0.57 0.23 0.67 A317RNA RV01 (10) 0.07 0.10 0.13 1.30 0.59 1.32 RV01 (08) 0.02 0.04 0.060.46 0.30 0.51 RV01 (05) 0.08 0.12 0.15 1.90 0.68 1.94 RV01 (09) 0.060.08 0.09 1.62 0.67 1.71 RV01 (10) 0.03 0.08 DNA RV13 (02) 0.03 0.040.06 1.15 0.41 1.18

Thus the liposome formulations significantly enhanced immunogenicityrelative to the naked RNA controls, as determined by increasedF-specific IgG titers and T cell frequencies. Plasmid DNA formulatedwith liposomes, or delivered naked using electroporation, wassignificantly less immunogenic than liposome-formulated self-replicatingRNA.

The RV01 RNA vaccines were more immunogenic than the RV13 vaccine. RV01has a tertiary amine in the headgroup with a pKa of about 5.8, and alsoinclude unsaturated alkyl tails. RV13 has unsaturated alkyl tails butits headgroup has a quaternary amine and is very strongly cationic.

Liposomes—Requirement for Encapsulation

To assess whether the effect seen in the liposome groups was due merelyto the liposome components, or was linked to the encapsulation, thereplicon was administered in encapsulated form (with two differentpurification protocols, 0.1 μg RNA), or mixed with the liposomes aftertheir formation (a non-encapsulated “lipoplex”, 0.1 μg RNA), or as nakedRNA (1 μg). FIG. 10 shows that the lipoplex gave the lowest levels ofexpression, showing that shows encapsulation is essential for potentexpression.

Further experiments used three different RNAs: (i) ‘vA317’ replicon thatexpresses RSV-F i.e. the surface fusion glycoprotein of RSV; (ii) ‘vA17’replicon that expresses GFP; and (iii) ‘vA336’ that isreplication-defective and encodes GFP.

RNAs were delivered either naked or with liposomes made by method (D).Empty liposomes were made by method (D) but without any RNA. Fourliposome formulations had these characteristics:

RNA Particle Size Zav (nm) Polydispersity RNA Encapsulation vA317 155.70.113 86.6% vA17 148.4 0.139  92% vA336 145.1 0.143 92.9% Empty 147.90.147 —

BALB/c mice, 5 animals per group, were given bilateral intramuscularvaccinations (50 μL per leg) on days 0 and 21 with:

-   -   Group 1 naked self-replicating RSV-F RNA (vA317, 0.1 μg)    -   Group 2 self-replicating RSV-F RNA (vA317, 0.1 μg) encapsulated        in liposomes    -   Group 3 self-replicating RSV-F RNA (vA317, 0.1 μg) added to        empty liposomes    -   Group 4 F subunit protein (5 μg)

Serum was collected for antibody analysis on days 14, 35 and 51.F-specific specific serum IgG titers (GMT) were measured; if anindividual animal had a titer of <25 (limit of detection), it wasassigned a titer of 5. In addition, spleens were harvested from mice atday 51 for T cell analysis, to determine cells which werecytokine-positive and specific for RSV F51-66 peptide (CD4+) or for RSVF peptides F85-93 and F249-258 (CD8+).

IgG titers were as follows in the 10 groups and in non-immunised controlmice:

Day 1 2 3 4 — 14 22 1819 5 5 5 35 290 32533 9 19877 5 51 463 30511 1820853 5

RSV serum neutralization titers at day 51 were as follows:

Day 1 2 3 4 51 35 50 24 38

Animals showing RSV F-specific CD4+ splenic T cells on day 51 were asfollows, where a number (% positive cells) is given only if thestimulated response was statistically significantly above zero:

Cytokine 1 2 3 4 IFN-γ 0.04 IL2 0.02 0.06 0.02 IL5 TNFα 0.03 0.05

Animals showing RSV F-specific CD8+ splenic T cells on day 51 were asfollows, where a number is given only if the stimulated response wasstatistically significantly above zero:

Cytokine 1 2 3 4 IFN-γ 0.37 0.87 IL2 0.11 0.40 0.04 IL5 TNFα 0.29 0.790.06

Thus encapsulation of RNA within the liposomes is necessary for highimmunogenicity, as a simple admixture of RNA and the liposomes (group 3)was not immunogenic (in fact, less immunogenic than naked RNA).

In other studies mice received various combinations of (i)self-replicating RNA replicon encoding full-length RSV F protein (ii)self-replicating GFP-encoding RNA replicon (iii) GFP-encoding RNAreplicon with a knockout in nsP4 which eliminates self-replication (iv)full-length RSV F-protein. 13 groups in total received:

A — — B 0.1 μg of (i), naked — C 0.1 μg of (i), encapsulated — inliposome D 0.1 μg of (i), with separate — liposomes E 0.1 μg of (i),naked 10 μg of (ii), naked F 0.1 μg of (i), naked 10 μg of (iii), nakedG 0.1 μg of (i), encapsulated 10 μg of (ii), naked in liposome H 0.1 μgof (i), encapsulated 10 μg of (iii), naked in liposome I 0.1 μg of (i),encapsulated 1 μg of (ii), encapsulated in liposome in liposome J 0.1 μgof (i), encapsulated 1 μg of (iii), encapsulated in liposome in liposomeK 5 μg F protein — L 5 μg F protein 1 μg of (ii), encapsulated inliposome M 5 μg F protein 1 μg of (iii), encapsulated in liposome

Results in FIG. 16 show that F-specific IgG responses requiredencapsulation in the liposome rather than mere co-delivery (comparegroups C & D). A comparison of groups K, L and M shows that the RNAprovided an adjuvant effect against co-delivered protein, and thiseffect was seen with both replicating and non-replicating RNA.

RSV Immunogenicity in Different Mouse Strains

Replicon “vA142” encodes the full-length wild type surface fusion (F)glycoprotein of RSV but with the fusion peptide deleted, and the 3′ endis formed by ribozyme-mediated cleavage. It was tested in threedifferent mouse strains.

BALB/c mice were given bilateral intramuscular vaccinations (50 μL perleg) on days 0 and 22. Animals were divided into 8 test groups (5animals per group) and a naïve control (2 animals):

-   -   Group 1 were given naked replicon (1 μg).    -   Group 2 were given 1 μg replicon delivered in liposomes        “RV01(37)” with 40% DlinDMA, 10% DSPC, 48% Chol, 2%        PEG-conjugated DMG.    -   Group 3 were given the same as group 2, but at 0.1 μg RNA.    -   Group 4 were given 1 μg replicon in “RV17(10)” liposomes (40%        RV17 (see above), 10% DSPC, 49.5% cholesterol, 0.5% PEG-DMG).    -   Group 5 were 1 μg replicon in “RV05(11)” liposomes (40% RV07        lipid, 30% 18:2 PE (DLoPE, 28% cholesterol, 2% PEG-DMG).    -   Group 6 were given 0.1 μg replicon in “RV17(10)” liposomes.    -   Group 7 were given 5 μg RSV-F subunit protein adjuvanted with        aluminium hydroxide.    -   Group 8 were a naïve control (2 animals)

Sera were collected for antibody analysis on days 14, 35 and 49.F-specific serum IgG GMTs were:

Day 1 2 3 4 5 6 7 8 14 82 2463 1789 2496 1171 1295 1293 5 35 1538 3418125605 23579 13718 8887 73809 5

At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows:

IgG 1 2 3 4 5 6 7 IgG1 94 6238 4836 7425 8288 1817 78604 IgG2a 538677064 59084 33749 14437 17624 24

RSV serum neutralizing antibody titers at days 35 and 49 were as follows(data are 60% plaque reduction neutralization titers of pools of 2-5mice, 1 pool per group):

Day 1 2 3 4 5 6 7 8 35 <20 143 20 101 32 30 111 <20 49 <20 139 <20 83 4132 1009 <20

Spleens were harvested at day 49 for T cell analysis. Average netF-specific cytokine-positive T cell frequencies (CD4+ or CD8+) were asfollows, showing only figures which were statistically significantlyabove zero (specific for RSV peptides F51-66, F164-178, F309-323 forCD4+, or for peptides F85-93 and F249-258 for CD8+):

CD4+CD8− CD4−CD8+ Group IFNγ IL2 IL5 TNFα IFNγ IL2 IL5 TNFα 1 0.03 0.060.08 0.47 0.29 0.48 2 0.05 0.10 0.08 1.35 0.52 1.11 3 0.03 0.07 0.060.64 0.31 0.61 4 0.05 0.09 0.07 1.17 0.65 1.09 5 0.03 0.08 0.07 0.650.28 0.58 6 0.05 0.07 0.07 0.74 0.36 0.66 7 0.02 0.04 0.04 8

C57BL/6 mice were immunised in the same way, but a 9th group receivedVRPs (1×10⁶ IU) expressing the full-length wild-type surface fusionglycoprotein of RSV (fusion peptide deletion).

Sera were collected for antibody analysis on days 14, 35 & 49.F-specific IgG titers (GMT) were:

Day 1 2 3 4 5 6 7 8 9 14 1140 2133 1026 2792 3045 1330 2975 5 1101 351721 5532 3184 3882 9525 2409 39251 5 12139

At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows:

IgG 1 2 3 4 5 6 7 8 IgG1 66 247 14 328 468 92 56258 79 IgG2a 2170 76855055 6161 1573 2944 35 14229

RSV serum neutralizing antibody titers at days 35 and 49 were as follows(data are 60% plaque reduction neutralization titers of pools of 2-5mice, 1 pool per group):

Day 1 2 3 4 5 6 7 8 9 35 <20 27 29 22 36 <20 28 <20 <20 49 <20 44 30 2336 <20 33 <20 37

Spleens were harvested at day 49 for T cell analysis. Average netF-specific cytokine-positive T cell frequencies (CD8+) were as follows,showing only figures which were statistically significantly above zero(specific for RSV peptides F85-93 and F249-258):

CD4−CD8+ Group IFNγ IL2 IL5 TNFα 1 0.42 0.13 0.37 2 1.21 0.37 1.02 31.01 0.26 0.77 4 1.26 0.23 0.93 5 2.13 0.70 1.77 6 0.59 0.19 0.49 7 0.100.05 8 9 2.83 0.72 2.26

Nine groups of C3H/HeN mice were immunised in the same way. F-specificIgG titers (GMT) were:

Day 1 2 3 4 5 6 7 8 9 14 5 2049 1666 1102 298 984 3519 5 806 35 15227754 19008 17693 3424 6100 62297 5 17249

At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows:

IgG 1 2 3 4 5 6 7 8 IgG1 5 1323 170 211 136 34 83114 189 IgG2a 302136941 78424 67385 15667 27085 3800 72727

RSV serum neutralizing antibody titers at days 35 and 49 were asfollows:

Day 1 2 3 4 5 6 7 8 9 35 <20 539 260 65 101 95 443 <20 595 49 <20 456296 35 82 125 1148 <20 387

Thus three different lipids (RV01, RV05, RV17; pKa 5.8, 5.85, 6.1) weretested in three different inbred mouse strains. For all 3 strains RV01was more effective than RV17; for BALB/c and C3H strains RV05 was lesseffective than either RV01 or RV17, but it was more effective in B6strain. In all cases, however, the liposomes were more effective thantwo cationic nanoemulsions which were tested in parallel.

CMV Immunogenicity

RV01 liposomes with DLinDMA as the cationic lipid were used to deliverRNA replicons encoding cytomegalovirus (CMV) glycoproteins. The “vA160”replicon encodes full-length glycoproteins H and L (gH/gL), whereas the“vA322” replicon encodes a soluble form (gHsol/gL). The two proteins areunder the control of separate subgenomic promoters in a single replicon;co-administration of two separate vectors, one encoding gH and oneencoding gL, did not give good results.

BALB/c mice, 10 per group, were given bilateral intramuscularvaccinations (50 μL per leg) on days 0, 21 and 42 with VRPs expressinggH/gL (1×10⁶ IU), VRPs expressing gHsol/gL (1×10⁶ IU) and PBS as thecontrols. Two test groups received 1 μg of the vA160 or vA322 repliconformulated in liposomes (40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-DMG;made using method (D) but with 150 μg RNA batch size).

The vA160 liposomes had a Zav diameter of 168 nm, a pdI of 0.144, and87.4% encapsulation. The vA322 liposomes had a Zav diameter of 162 nm, apdI of 0.131, and 90% encapsulation.

The replicons were able to express two proteins from a single vector.

Sera were collected for immunological analysis on day 63 (3wp3). CMVneutralization titers (the reciprocal of the serum dilution producing a50% reduction in number of positive virus foci per well, relative tocontrols) were as follows:

gH/gL VRP gHsol/gL VRP gH/gL liposome gHsol/gL liposome 4576 2393 424010062

RNA expressing either a full-length or a soluble form of the CMV gH/gLcomplex thus elicited high titers of neutralizing antibodies, as assayedon epithelial cells. The average titers elicited by theliposome-encapsulated RNAs were at least as high as for thecorresponding VRPs.

Repeat experiments confirmed that the replicon was able to express twoproteins from a single vector. The RNA replicon gave a 3wp3 titer of11457, compared to 5516 with VRPs.

Further experiments used different replicons in addition to vA160. ThevA526 replicon expresses the CMV pentameric complex(gH-gL-UL128-UL130-UL-131) under the control of three subgenomicpromoters: the first drives the expression of gH; the second drivesexpression of gL; the third drives the expression of theUL128-2A-UL130-2A-UL131 polyprotein, which contains two 2A cleavagesites between the three UL genes. The vA527 replicon expresses the CMVpentameric complex via three subgenomic promoters and two IRESs: thefirst subgenomic promoter drives the expression of gH; the secondsubgenomic promoter drives expression of gL; the third subgenomicpromoter drives the expression of the UL128; UL130 is under the controlof an EMCV IRES; UL131 is under control of an EV71 IRES. These threereplicons were delivered by liposome (method (H), with 150 μg batchsize) or by VRPs.

BALB/c mice, 10 groups of 10 animals, were given bilateral intramuscularvaccinations (50 μL per leg) on days 0, 21 and 42 with:

-   -   Group 1 VRPs expressing gH FL/gL (1×10⁶ IU)    -   Group 2 pentameric, 2A VRP (1×10⁵ IU)    -   Group 3 pentameric, 2A VRP (1×10⁶ IU)    -   Group 4 pentameric, IRES VRP (1×10⁵ IU)    -   Group 5 self-replicating RNA vA160 (l μg) formulated in        liposomes    -   Group 6 self-replicating RNA vA526 (1 μg) formulated in        liposomes    -   Group 7 self-replicating RNA vA527 (1 μg) formulated in        liposomes    -   Group 8 self-replicating RNA vA160 (1 μg) formulated in a        cationic nanoemulsion    -   Group 9 self-replicating RNA vA526 (1 μg) formulated in a        cationic nanoemulsion    -   Group 10 self-replicating RNA vA527 (1 μg) formulated in a        cationic nanoemulsion.

Sera were collected for immunological analysis on days 21 (3wp1), 42(3wp2) and 63 (3wp3).

CMV serum neutralization titers on days 21, 42 and 63 were:

Vaccine Group 3wp1 3wp2 3wp3 1 126 6296 26525 2 N/A N/A 6769 3 N/A 34427348 4 N/A N/A 2265 5 347 9848 42319 6 179 12210  80000 7 1510  51200 130000 8 N/A N/A 845 9 N/A N/A 228 10 N/A N/A 413

Thus self-replicating RNA can be used to express multiple antigens froma single vector and to raise a potent and specific immune response. Thereplicon can express five antigens (CMV pentamric complex(gH-gL-UL128-UL130-UL-131) and raise a potent immune response.Self-replicating RNA delivered in liposomes was able to elicit hightiters of neutralizing antibody, as assayed on epithelial cells, at alltime points assayed (3wp1, 3wp2, and 3wp3). These responses weresuperior to the corresponding VRPs and to cationic nanoemulsions.

Delivery Volume

Hydrodynamic delivery employs the force generated by the rapid injectionof a large volume of solution to overcome the physical barriers of cellmembranes which prevent large and membrane-impermeable compounds fromentering cells. This phenomenon has previously been shown to be usefulfor the intracellular delivery of DNA vaccines.

A typical mouse delivery volume for intramuscular injection is 50 μlinto the hind leg, which is a relatively high volume for a mouse legmuscle. In contrast, a human intramuscular dose of ˜0.5 ml is relativelysmall. If immunogenicity in mice would be volume-dependent then thereplicon vaccines' efficacy might be due, at least in part, onhydrodynamic forces, which would not be encouraging for use of the samevaccines in humans and larger animals.

The vA317 replicon was delivered to BALB/c mice, 10 per group, bybilateral intramuscular vaccinations (5 or 50 per leg) on day 0 and 21:

-   -   Group 1 received naked replicon, 0.2 μg in 50 μL per leg    -   Group 2 received naked replicon, 0.2 μg in 5 μL per leg    -   Group 3 received liposome-formulated replicon (0.2 μg, 50 μL per        leg)    -   Group 4 received liposome-formulated replicon (0.2 μg, 5 μL per        leg)

Serum was collected for antibody analysis on days 14 and 35. F-specificserum IgG GMTs were:

Day 1 2 3 4 14 42 21 2669 2610 35 241 154 17655 18516

Thus immunogenicity of the formulated replicon did not vary according tothe delivered volume, thus indicating that these RNA vaccines do notrely on hydrodynamic delivery for their efficacy.

Expression Kinetics

A self-replicating RNA replicon (“vA311”) that expresses a luciferasereporter gene (luc) was used for studying the kinetics of proteinexpression after injection. BALB/c mice, 5 animals per group, receivedbilateral intramuscular vaccinations (50 μL per leg) on day 0 with:

-   -   Group 1 DNA expressing luciferase, delivered using        electroporation (10 μg)    -   Group 2 self-replicating RNA (1 μg) formulated in liposomes    -   Group 3 self-replicating RNA (1 μg) formulated with a cationic        nanoemulsion    -   Group 4 self-replicating RNA (1 μg) formulated with a cationic        nanoemulsion    -   Group 5 VRP (1×10⁶ IU) expressing luciferase

Prior to vaccination mice were depilated. Mice were anesthetized (2%isoflurane in oxygen), hair was first removed with an electric razor andthen chemical Nair. Bioluminescence data was then acquired using aXenogen IVIS 200 imaging system (Caliper Life Sciences) on days 3, 7,14, 21, 28, 35, 42, 49, 63 and 70. Five minutes prior to imaging micewere injected intraperitoneally with 8 mg/kg of luciferin solution.Animals were then anesthetized and transferred to the imaging system.

Image acquisition times were kept constant as bioluminescence signal wasmeasured with a cooled CCD camera.

In visual terms, luciferase-expressing cells were seen to remainprimarily at the site of RNA injection, and animals imaged after removalof quads showed no signal.

In quantitative terms, luciferase expression was measured as averageradiance over a period of 70 days (p/s/cm²/sr), and results were asfollows for the 5 groups:

Days 1 2 3 4 5 3 8.69E+07 3.33E+06 2.11E+06 9.71E+06 1.46E+07 7 1.04E+088.14E+06 1.83E+07 5.94E+07 1.64E+07 14 8.16E+07 2.91E+06 9.22E+063.48E+07 8.49E+05 21 1.27E+07 3.13E+05 6.79E+04 5.07E+05 6.79E+05 281.42E+07 6.37E+05 2.36E+04 4.06E+03 2.00E+03 35 1.21E+07 6.12E+052.08E+03 42 1.49E+07 8.70E+05 49 1.17E+07 2.04E+05 63 9.69E+06 1.72E+0370 9.29E+06

The self-replicating RNA formulated with cationic nanoemulsions showedmeasurable bioluminescence at day 3, which peaked at day 7 and thenreduced to background levels by days 28 to 35. When formulated inliposomes the RNA showed measurable bioluminescence at day 3, whichpeaked at day 7 and reduced to background levels by day 63. RNAdelivered using VRPs showed enhanced bioluminescence at day 21 whencompared to the formulated RNA, but expression had reduced to backgroundlevels by day 28. Electroporated DNA showed the highest level ofbioluminescence at all time points measured and levels ofbioluminescence did not reduce to background levels within the 70 daysof the experiment.

Delivery Route

Liposome-encapsulated RNA encoding HIV gp140 was delivered to miceintramuscularly, intradermally, or subcutaneously. All three routes ledto high serum IgG levels of HIV-specific antibodies (FIG. 15), exceedingtiters seen in response to electroporated intramuscular DNA.

Cotton Rats

A study was performed in cotton rats (Sigmodon hispidis) instead ofmice. At a 1 μg dose liposome encapsulation increased F-specific IgGtiters by 8.3-fold compared to naked RNA and increased PRNT titers by9.5-fold. The magnitude of the antibody response was equivalent to thatinduced by 5×10⁶ IU VRP. Both naked and liposome-encapsulated RNA wereable to protect the cotton rats from RSV challenge (1×10⁵ plaque formingunits), reducing lung viral load by at least 3.5 logs. Encapsulationincreased the reduction by about 2-fold.

Further work in cotton rats used four different replicons: vA317expresses full-length RSV-F; vA318 expresses truncated (transmembraneand cytoplasmic tail removed) RSV-F; vA142 expresses RSV-F with itsfusion peptide deleted; vA140 expresses the truncated RSV-F also withoutits peptide. Cotton rats, 4 to 8 animals per group, were givenintramuscular vaccinations (100 μL in one leg) on days 0 and 21 with thefour different replicons at two doses (1.0 and 0.1 μg) formulated inliposomes made by method (D), but with a 150 μg RNA batch size. Controlgroups received a RSV-F subunit protein vaccine (5 μg) adjuvanted withalum (8 animals/group), VRPs expressing full-length RSV-F (1×10⁶ IU, 8animals/group), or naïve control (4 animals/group). Serum was collectedfor antibody analysis on days 0, 21 and 34.

F-specific serum IgG titers and RSV serum neutralizing antibody titerson day 21 and 34 were:

IgG, IgG, NT, NT, Group day 21 day 34 day 21 day 34 1 μg vA317 915 2249115 459 0.1 μg vA317 343 734 87 95 1 μg vA318 335 1861 50 277 0.1 μgvA318 129 926 66 239 1 μg vA142 778 4819 92 211 0.1 μg vA142 554 2549 78141 1 μg vA140 182 919 96 194 0.1 μg vA140 61 332 29 72 5 μg F trimersubunit/alum 13765 86506 930 4744 1 × 10⁶ IU VRP-F full 1877 19179 1044528 Naïve 5 5 10 15

All four replicons evaluated in this study (vA317, vA318, vA142, vA140)were immunogenic in cotton rats when delivered by liposome, althoughserum neutralization titers were at least ten-fold lower than thoseinduced by adjuvanted protein vaccines or by VRPs. The liposome/RNAvaccines elicited serum F-specific IgG and RSV neutralizing antibodiesafter the first vaccination, and a second vaccination boosted theresponse effectively. F-specific IgG titers after the second vaccinationwith 1 μg replicon were 2- to 3-fold higher than after the secondvaccination with 0.1 μg replicon. The four replicons elicited comparableantibody titers, suggesting that full length and truncated RSV-F, eachwith or without the fusion peptide, are similarly immunogenic in cottonrats.

Further work in cotton rats again used the vA317, vA318 and vA142replicons. Cotton rats, 2-8 animals per group, were given intramuscularvaccinations (100 μL in one leg) on days 0 and 21 with the replicons(0.1 or 1 μg) encapsulated in RV01 liposomes made by method (D) but witha 150 μg RNA batch size. Control groups received the RSV-F subunitprotein vaccine (5 μg) adjuvanted with alum or VRPs expressingfull-length RSV-F (1×10⁶ IU, 8 animals/group). All these animalsreceived a third vaccination (day 56) with RSV-F subunit protein vaccine(5 μg) adjuvanted with alum. In addition there was a naïve control (4animals/group). In addition, an extra group was given bilateralintramuscular vaccinations (50 μL per leg) on days 0 and 56 with 1 μgvA317 RNA in liposomes but did not receive a third vaccination with thesubunit protein vaccine.

Serum was collected for antibody analysis on days 0, 21, 35, 56, 70,plus days 14, 28 & 42 for the extra group. F-specific serum IgG titers(GMT) were as follows:

Day 21 Day 35 Day 56 Day 70 1 μg vA318 260 1027 332 14263 0.1 μg vA31895 274 144 2017 1 μg vA142 483 1847 1124 11168 0.1 μg vA142 314 871 41811023 1 μg vA317 841 4032 1452 13852 1 × 10⁶ VRP (F-full) 2075 3938 159614574 5 μg F trimer subunit/alum 12685 54526 25846 48864 Naïve 5 5 5 5

Serum neutralisation titers were as follows (60% RSV neutralizationtiters for 2 pools of 3-4 animals per group, GMT of these 2 pools pergroup):

Day 21 Day 35 Day 56 Day 70 1 μg vA318 58 134 111 6344 0.1 μg vA318 41102 63 6647 1 μg vA142 77 340 202 5427 0.1 μg vA142 35 65 56 2223 1 μgvA317 19 290 200 4189 1 × 10⁶ VRP (F-full) 104 1539 558 2876 5 μg Ftrimer subunit/alum 448 4457 1630 3631 Naïve 10 10 10

Serum titers and neutralising titers for the extra group were asfollows:

Day 14 21 28 35 42 56 70 IgG 397 561 535 501 405 295 3589 NT 52 82 90106 80 101 1348

Thus the replicons are confirmed as immunogenic in cotton rats,eliciting serum F-specific IgG and RSV neutralizing antibodies after thefirst vaccination. A second vaccination boosted the responseseffectively. F-specific IgG titers after the second vaccination with 1.0μg replicon were 1.5 to 4-fold higher than after the second vaccinationwith 0.1 μg replicon.

The third vaccination (protein at day 56) did not boost titers in cottonrats previously vaccinated with F trimer subunit+alum, but it didprovide a large boost to titers in cotton rats previously vaccinatedwith replicon. In most cases the RSV serum neutralization titers aftertwo replicon vaccinations followed by protein boost were equal to orgreater than titers induced by two or three sequential proteinvaccinations.

This study also evaluated the kinetics of the antibody response to 1.0μg vA317. F-specific serum IgG and RSV neutralization titers induced bya single vaccination reached their peak around day 21 and weremaintained through at least day 56 (50-70% drop in F-specific IgG titer,little change in RSV neutralization titer). A homologous secondvaccination was given to these animals on day 56, and boosted antibodytiters to a level at least equal to that achieved when the secondvaccination was administered on day 21.

Further experiments involved a viral challenge. The vA368 repliconencodes the full-length wild type surface fusion glycoprotein of RSVwith the fusion peptide deleted, with expression driven by the EV71IRES. Cotton rats, 7 per group, were given intramuscular vaccinations(100 μL per leg) on days 0 and 21 with vA368 in liposomes prepared bymethod (H), 175 μg RNA batch size, or with VRPs having the samereplicon. A control group received 5 μg alum-adjuvanted protein, and anaïve control group was also included.

All groups received an intranasal challenge (i.n.) with 1×10⁶ PFU RSVfour weeks after the final immunization. Serum was collected forantibody analysis on days 0, 21, 35. Viral lung titers were measured 5days post challenge. Results were as follows:

Liposome VRP Protein Naïve F-specific Serum IgG titers (GMT) Day 21 3701017 28988 5 Day 35 2636 2002 113843 5 Neutralising titers (GMT) Day 2147 65 336 10 Day 35 308 271 5188 10 Lung viral load (pfu per gram oflung) Day 54 422 225 124 694110

Thus the RNA vaccine reduced the lung viral load by over three logs,from approximately 10⁶ PFU/g in unvaccinated control cotton rats to lessthan 10³ PFU/g in vaccinated cotton rats.

Large Mammal Study

A large-animal study was performed in cattle. Calves (4-6 weeks old,˜60-80 kg, 5 per group) were immunised with 66 μg of replicon vA317encoding full-length RSV F protein at days 0, 21, 86 and 146. Thereplicons were formulated inside liposomes. PBS alone was used as anegative control, and a licensed vaccine was used as a positive control(“Triangle 4” from Fort Dodge, containing killed virus). All calvesreceived 15 μg F protein adjuvanted with the MF59 emulsion on day 146.One cow was mistakenly vaccinated with the wrong vaccine on day 86instead of Triangle 4 and so its data were excluded from day 100onwards.

The RNA vaccines encoded human RSV F whereas the “Triangle 4” vaccinecontains bovine RSV F, but the RSV F protein is highly conserved betweenBRSV and HRSV.

The liposomes were made by method (E), except a 1.5 mg RNA batch sizewas used.

Calves received 2 ml of each experimental vaccine, administeredintramuscularly as 2×1 ml on each side of the neck. In contrast, the“Triangle 4” vaccine was given as a single 2 ml dose in the neck.

Serum was collected for antibody analysis on days 0, 14, 21, 35, 42, 56,63, 86, 100, 107, 114, 121, 128, 135, 146, 160, 167, 174, 181, 188, 195,and 202. If an individual animal had a titer below the limit ofdetection it was assigned a titer of 5

FIG. 14A shows F-specific IgG titers over the first 63 days. The RNAreplicon was immunogenic in the cows via liposomes, although it gavelower titers than the licensed vaccine. All vaccinated cows showedF-specific antibodies after the second dose, and titers were very stablefrom the period of 2 to 6 weeks after the second dose (and wereparticularly stable for the RNA vaccines).

FIG. 14B shows F-specific serum IgG titers (GMT) over 210 days, andmeasured values up to day 202 were as follows:

3wp1 2wp2 5wp2 ~9wp2 2wp3 5wp3 8wp3 2wp4 5wp4 8wp4 D 0 D 21 D 35 D 56 D86 D 100 D 121 D 146 D 160 D 181 D 202 PBS 5 5 5 5 5 5 5 5 46 98 150Liposome 5 5 12 11 20 768 428 74 20774 7022 2353 Triangle 4 5 5 1784 721514 3406 2786 336 13376 4775 2133

RSV serum neutralizing antibody titers were as follows:

2wp2 5wp2 2wp3 3wp3 4wp3 8wp3 2wp4 3wp4 4wp4 D 0 D 35 D 56 D 100 D 107 D114 D 146 D 160 D 167 D 174 PBS 12 10 10 14 18 20 14 10 10 10 Liposome13 10 10 20 13 17 13 47 26 21 Triangle 4 12 15 13 39 38 41 13 24 26 15

The material used for the second liposome dose was not freshly prepared,and the same lot of RNA showed a decrease in potency in a mouseimmunogenicity study. Therefore it is possible that the vaccine wouldhave been more immunogenic if fresh material had been used for allvaccinations.

When assayed with complement, neutralizing antibodies were detected inall vaccinated cows. In this assay, all vaccinated calves had goodneutralizing antibody titers after the second RNA vaccinationFurthermore, the RNA vaccine elicited F-specific serum IgG titers thatwere detected in a few calves after the second vaccination and in allcalves after the third.

MF59-adjuvanted RSV-F was able to boost the IgG response in allpreviously vaccinated calves, and to boost complement-independentneutralization titers of calves previously vaccinated with RNA.

Proof of concept for RNA vaccines in large animals is particularlyimportant in light of the loss in potency observed previously withDNA-based vaccines when moving from small animal models to largeranimals and humans. A typical dose for a cow DNA vaccine would be 0.5-1mg [47,48] and so it is very encouraging that immune responses wereinduced with only 66 μg of RNA.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

TABLE 1 useful phospholipids DDPC1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA1,2-Dierucoyl-sn-Glycero-3-Phosphate DEPC1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanol- amine DEPG1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DLOPC1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine DLPA1,2-Dilauroyl-sn-Glycero-3-Phosphate DLPC1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine DLPE1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanol- amine DLPG1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DLPS1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine DMG1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine DMPA1,2-Dimyristoyl-sn-Glycero-3-Phosphate DMPC1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine DMPE1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanol- amine DMPG1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DMPS1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine DOPA1,2-Dioleoyl-sn-Glycero-3-Phosphate DOPC1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine DOPE1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanol- amine DOPG1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DOPS1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine DPPA1,2-Dipalmitoyl-sn-Glycero-3-Phosphate DPPC1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine DPPE1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanol- amine DPPG1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DPPS1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine DPyPE1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine DSPA1,2-Distearoyl-sn-Glycero-3-Phosphate DSPC1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine DSPE1,2-Diostearpyl-sn-Glycero-3-phosphatidylethanol- amine DSPG1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DSPS1,2-Distearoyl-sn-Glycero-3-phosphatidylserine EPC Egg-PC HEPCHydrogenated Egg PC HSPC High purity Hydrogenated Soy PC HSPCHydrogenated Soy PC LYSOPC 1-Myristoyl-sn-Glycero-3-phosphatidylcholineMYRISTIC LYSOPC 1-Palmitoyl-sn-Glycero-3-phosphatidylcholine PALMITICLYSOPC 1-Stearoyl-sn-Glycero-3-phosphatidylcholine STEARIC Milk1-Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidyl- Sphingomyelin cholineMPPC MSPC 1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidyl- choline PMPC1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidyl- choline POPC1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidyl- choline POPE1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidyl- ethanolamine POPG1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol) . . .] PSPC1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidyl- choline SMPC1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidyl- choline SOPC1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidyl- choline SPPC1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidyl- choline

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The invention claimed is:
 1. A non-virion particle for in vivo deliveryof RNA to a vertebrate cell, said particle comprising a deliverymaterial and a self-replicating RNA molecule that encodes a polypeptideimmunogen, wherein (a) said non-virion particle does not comprise aprotein capsid, said delivery material is a liposome, and the RNA isencapsulated in the liposome (a “LNP”); (b) the RNA molecule does notcomprise modified nucleotides, other than a 5′cap, is not packaged withstructural proteins as a virion, and cannot induce production ofRNA-containing virions; (c) said non-virion particle can deliver RNA toa vertebrate cell in vivo; and (d) wherein the immunogen is translatedin vivo and can elicit an immune response against said immunogen.
 2. Theparticle of claim 1, wherein the self-replicating RNA molecule encodes(i) a RNA-dependent RNA polymerase which can transcribe RNA from theself-replicating RNA molecule and (ii) an immunogen.
 3. The particle ofclaim 2, wherein the RNA molecule comprises two open reading frames, thefirst of which encodes an alphavirus replicase and the second of whichencodes the immunogen.
 4. The particle of claim 1, wherein the RNAmolecule is 9000-12000 nucleotides long.
 5. The particle of claim 1,wherein the immunogen can elicit an immune response in vivo against abacterium, a virus, a fungus or a parasite.
 6. The particle of claim 5,wherein the immunogen can elicit an immune response in vivo againstrespiratory syncytial virus glycoprotein F.
 7. A pharmaceuticalcomposition comprising a particle of claim 1.